Shoot Base Research Articles

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

BackgroundMicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures.ResultsUsing in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots.ConclusionsThis is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2126-8) contains supplementary material, which is available to authorized users.

Polyamines and methyl jasmonate in bulb formation of in vitro propagated tulips

Effects of polyamines (PAs), their precursors, ornithine (Orn) and arginine (Arg), as well as methyl jasmonate (MeJA) were studied on bulb formation in vitro with four tulip cultivars differing in bulbing ability. The endogenous PA profiles of the shoots and bulbing shoots were monitored during successive micropropagation stages: S1, sixth week of the standard multiplication subculture; S2, fifth week of the last multiplication subculture; S3, 5 weeks later, just before the cold treatment; S4, at the end of the 13-week-cold treatment; and S5, 6 weeks after the end of cold treatment (shoot base swelling/bulb formation phase). Putrescine (Put), spermidine (Spd) and spermine (Spm) (50 and 100 µM) as well as Arg and Orn (500 and 1000 µM) were added to the medium at various bulbing stages (S3, S4 and S5). Positive effects of these compounds on bulb formation were found for the cultivar ‘New Beet’ (NB), with the lowest bulbing ability. Both PAs and their precursors, when used at lower concentrations at the beginning of bulbing process (S3), together with standard MeJA treatment, significantly enhanced bulb numbers in this cultivar. Putrescine was the major endogenous polyamine at the stages of shoot multiplication (S1–S2). Spd levels were the highest in ‘Fringed Black’ (FB), the cultivar of extremely high bulbing ability, and this PA dominated over Put during the entire micropropagation process in this cultivar. Endogenous Spd contents manifested similar dynamics in all cultivars showing two peaks: at shoot multiplication (S2) and at the end of cooling (S4). The contents of all the PAs were the lowest at the final bulbing stage (S5) except the Spm level in cultivar NB in which it increased at this time. It is postulated that the high bulbing competence may be related to a transient increase in Spd content in response to low temperature treatment (S4), followed by its rapid decrease during bulb growth and maturation (S5, 6 weeks after the end of cooling) when developing bulbs begin to enter dormancy. The low levels of all the PAs at final bulbing stage as well as low Put/Spd ratio may also aid in the bulb formation of tulip in vitro.

Direct adventitious shoot regeneration, in vitro flowering, fruiting, secondary metabolite content and antioxidant activity of Scrophularia takesimensis Nakai

Effects of plant growth regulators, light quality and sucrose on direct adventitious shoot regeneration, in vitro flowering and fruiting of Scrophularia takesimensis were investigated. The highest mean number of shoots per leaf (23.4), petiole (17.2) and stem (20.3) explants was obtained on Murashige and Skoog (MS) medium fortified with 2.0 mg l−1 BA and 1.0 mg l−1 IAA under white fluorescent light (WFL). Red light emitting diodes (LED) gave better shoot growth followed by WFL and blue LED. However, red LED treatment decreased the number of roots and induced callus at the base of shoot. The highest number of roots per shoot (9.2) and a maximum root length (8.9 cm) were obtained when the cultures were maintained under WFL. Somaclonal variation was observed when the shoot buds were cultured on the modified MS medium containing 1.0 mg l−1 IBA. The variant had variegated leaves in comparison with those of normal plantlets. The greatest frequency of flower induction (96.8 %) was obtained when the shoots were cultured on the modified MS medium containing 6.0 % (w/v) sucrose for 45 days under blue LED. The in vitro developed flowers self-fertilized and formed fruits. The culture media and environment had a positive effect on the content of phenolic compounds. Harpagoside content was high in seeds and low in shoots developed in MS medium containing BA. Among the light sources, shoots maintained under blue LED found to be best for harpagoside production (4.9 mg g−1) followed by WFL (3.3 mg g−1) and red LED (2.7 mg g−1). In vitro regenerated shoots had higher capacity to detoxify DPPH free radicals than field-grown plant samples.

Morphology and Accumulation of Epicuticular Wax on Needles of Douglas-fir (Pseudotsuga menziesiivar.menziesii)

Abstract Past studies have documented differences in epicuticular wax among several tree species but little attention has been paid to changes in accumulation of foliar wax that can occur during the year. We sampled current-year needles from the terminal shoots of Douglas-fir (Pseudotsuga menziesii var. menziesii) in late June/early July, late August and early November. Needles were sampled from two sites that differed in their climate and shoot phenology. Adaxial (upper), abaxial (lower) and cross-sectional surfaces were examined on scanning electron micrographs. Wax thickness increased significantly (P < 0.01) during the year (from 2.9 ± 0.26 µm in late June/early July to 4.4 ±0.13 µm in early November). Mean wax thickness was slightly thicker on adaxial (4.0 ± 0.16 µm) than on abaxial (3.5 ± 0.22 µm) surfaces (P = 0.03). There were no significant differences in wax thickness between needles sampled at the base of the terminal shoot or near the tip of the shoot. Tubular or rod-shaped epicuticular wax cr...

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500mg l(-1) of IBA (indole-3 butyric acid) for 3min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85% survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

ORIGEM DE BROTAÇÕES EPICÓRMICAS E APLICAÇÃO DE ÁCIDO INDOLILBUTÍRICO NO ENRAIZAMENTO DE ESTACAS DE &lt;i&gt;Vochysia bifalcata&lt;/i&gt; Warm.

http://dx.doi.org/10.5902/1980509818457A guaricica (Vochysia bifalcata) é uma espécie nativa da região litorânea do Estado do Paraná, Santa Catarina, São Paulo, Rio de Janeiro e Minas Gerais, com grande importância ecológica, devido à sua capacidade para regenerar áreas degradadas. No entanto, existem poucas informações sobre a sua propagação, seja sexuada ou assexuada. Uma das causas do baixo enraizamento adventício de estacas caulinares é a maturação dos tecidos coletados para tal fim. Desta forma, o presente trabalho objetivou avaliar diferentes origens de brotações epicórmicas associadas à aplicação de ácido indolilbutírico (AIB) na propagação vegetativa de estacas semilenhosas de Vochysia bifalcata. Foram utilizados dois tipos de estacas (provenientes de brotação de decepa e envergadura de caule) nas quais foram aplicadas duas concentrações de AIB (0 e 1000 mg L-1). Após o plantio em tubetes com vermiculita de granulometria média e casca de arroz carbonizada, as estacas foram mantidas em casa de vegetação climatizada por 60 dias. Análises anatômicas da base de cada tipo de estaca demonstraram que a presença de fibras na região cortical do caule de estacas de decepa não constituiu uma barreira anatômica para a emissão radicial, pois estas apresentaram maior capacidade de enraizamento (81%), maior número de raízes por estaca (8,0) e maior comprimento das raízes (2,0 cm) em relação àquelas de envergadura de caule, que apresentaram resultados inferiores como porcentagem de enraizamento (31%), número de raízes por estaca (3,3) e comprimento das raízes (0,7 cm). Conclui-se, assim, que o uso de brotações epicórmicas induzidas por decepa de caule proporciona melhor enraizamento adventício do que de envergadura de caule, não necessitando da aplicação de AIB como indutor do enraizamento neste tipo de material.

A Proteomic Analysis of the Upper and Lower Flanks of the Base of Rice Shoot in the Gravitropism

Due to gravitational stimulation, the lower part of a shoot base grows faster than the upper part, leading the shoot to curve upward. Though much research has been done on the mechanism of plant gravitropism, it still requires extensive elucidation. Recently, functional genomic strategies have been applied to study this mechanism in plants. The present study carried out a proteomic analysis to gain a better understanding of gravity stimulation in rice. Three-week-old rice seedlings were gravitropically stimulated and samples were harvested at 4 different time points: 0.5, 3, 6, and 9 h. Then, the total crude proteins were extracted from the lower and upper parts of the shoot base, separated by 2-DE, and silver stained. At each time point, proteins in the lower and upper parts were compared, and the differently expressed proteins were identified using MALDI TOF or ESI-MS/MS. After gravity stimulation, proteins involved in nine different functional categories were either up-regulated or down-regulated. Sugar metabolism, glycolysis, the tricarboxylic acid (TCA/citric) cycle, pyruvate metabolism, and transcription regulation-related proteins were regulated. Although the initiation of defense reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues. Interestingly, the abundance of some proteins changed drastically at only 0.5 h after reorientation: inosine monophosphate dehydrogenase (up to 6.49-fold higher in lower flanks at 0.5 h), ATP synthase D (4.25-fold), and ribulose-1,5 -bisphosphate carboxylase oxygenase (3.62-fold). These findings may aid in understanding the mechanism of the gravitropism.

In vitro culture and production of syringin and rutin in Saussurea involucrata (Kar. et Kir.) - an endangered medicinal plant.

BackgroundSaussurea involucrata (Kar. et Kir.) commonly known as ‘snow lotus’ or ‘Xue Lian’ is an important plant in the traditional Chinese system of medicine. The plant contains flavonoids such as syringin and rutin. These compounds have been reported to be anti-rheumatic, anti-inflammatory and dilate blood vessels, lower blood pressure, prevent cardiovascular diseases, enhance immunity, and act as anti-aging, anti-cancer, and anti-fatigue agents. The species has become endangered due to the excessive collection of S. involucrata plants in the wild, slower plant growth and ecological destruction of natural habitats. There is a severe shortage of plant material, while the market demand is ever increasing. Hence, it is very important to apply tissue culture technique for plant propagation and production of the bioactive compounds of this species.ResultsMultiple shoot induction and proliferation in shoot base explants derived from in vitro raised seedlings of S. involucrata was achieved on 3/4 strength of Murashige and Skoog’s (MS) basal medium (MSBM) supplemented with 1.0 mg/L−1 BA and 1.5 mg/L−1 NAA. Rooting was induced in 100 % shoots cultured on 1/2X MSBM supplemented with 1.0 mg/L−1 IBA for one week and then transfer to auxin free medium. The plantlets could be acclimatized successfully by sachet technique and established in the greenhouse. Maximum callus induction and proliferation in leaf segments was achieved on 1/2X MSBM supplemented with 0.5 mg/L−1 BA, 0.5 mg/L−1 NAA, 0.4 % gelrite and on incubation at 20 °C. Container closures had an influence on the quality and quantity of callus and production of the active compounds. The HPLC analysis showed much higher syringin content in in vitro shoots and callus as compared to commercially available market crude drug.ConclusionThe present study describes an in vitro culture protocol of Saussurea involucrata. The bioactive compounds, syringin and rutin could be produced through tissue culture technique without sacrificing the endangered Saussurea involucrata plants in the wild.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-015-0092-8) contains supplementary material, which is available to authorized users.

Postcompression of high-energy terawatt-level femtosecond pulses and application to high-order harmonic generation

We perform a post-compression of high energy pulses by using optical-field ionization of low pressure helium gas in a guided geometry. We apply this approach to a TW chirped-pulse-amplification based Ti:Sapphire laser chain and show that spectral broadening can be controlled both with the input pulse energy and gas pressure. Under optimized conditions, we generate 10 fs pulses at TW level directly under vacuum and demonstrate a high stability of the post compressed pulse duration. These high energy post-compressed pulses are thereafter used to perform high harmonic generation in a loose focusing geometry. The XUV beam is characterized both spatially and spectrally on a single shot basis and structured continuous XUV spectra are observed.

Influence of the cryopreservation technique, recovery medium and genotype on genetic stability of mint cryopreserved shoot tips

This study compared the effect of the cryopreservation protocol, the genotype and the plant growth regulators used in the recovery media on the genetic stability of mint shoot tips. Shoot tips of two micropropagated genotypes of mint were cryopreserved either using an encapsulation-dehydration or droplet-vitrification protocol. Three recovery media were tested, all of them MS based although with different plant growth regulators: (1) 0.5 mg L−1 6-benzylaminopurine (BAP); (2) 0.5 mg L−1 6-dimethylallylamino-purine (2iP) + 0.1 mg L−1 α-naphthalene acetic acid (NAA); and (3) 0.5 mg L−1 BAP + 0.1 mg L−1 NAA. DNA was extracted from three different types of samples: leaves from shoots, callus at the base of shoots and callus. RAPD markers were used to assess the genetic stability. One of the genotypes, ‘MEN 198’, showed higher percentage of stable samples than the other one, ‘MEN 186’ (97 vs. 42 %; considering all media, protocol and type of explant). The material recovered after droplet-vitrification showed higher stability compared to the encapsulation-dehydration recovered samples: 99 versus 87 % in ‘MEN 198’, and 80 versus 24 % in ‘MEN 186’. There were not large differences in the genetic stability obtained from the three types of samples in ‘MEN 196’ (85–100 %), while in ‘MEN 186’ calli were more unstable (53, 44 and 30 % stability for leaves, basal callus and callus, respectively). The effect of the recovery medium on the stability of the samples was noticeable in the more unstable types of explants but not in leaves.

Auxin Type, Auxin Concentration, and Air and Substrate Temperature Difference Play Key Roles in the Rooting of Juvenile Hardwood Pecan Cuttings

This study examined the ability to vegetatively propagate 1-year-old pecan (Carya illinoinensis) through the rooting of hardwood cuttings. Cuttings were treated with varying concentrations of different auxins and different combinations of media and ambient temperatures. Under different temperature conditions, all auxin treatments induced the rooting of cuttings but did not promote sprouting. The effectiveness of the induction of adventitious roots was as follows: 1-naphthalene acetic acid (NAA) &gt; indole 3-butyric acid &gt; indole 3-acetic acid. The base of the parent shoot treated by NAA at a concentration of 0.09%, planted in substrate with bottom heat was the most effective, with 82% rooting, 8.3 roots/cutting and root lengths of 7.3 cm. These findings suggested that auxin and substrate/air temperature differences are both indispensable in the process of adventitious roots formation in pecan. This study revealed that the propagation of hardwood cuttings derived from branches of 1-year-old pecan is possible.

Multiplicação in vitro de Ipê-Amarelo (Handroanthus chrysotrichus)

This study aimed to evaluate the in vitro multiplication of ipe-amarelo tree in different 6- benzylaminopurine (BAP) concentrations. Nodal segments of seedlings obtained from the in vitro seeds germination were used as explants source being inoculated in nutrient broth ½ WPM containing different concentrations of BAP (0, 2, 4, 8 and 16 µM). The experiment was conducted in a completely randomized design with five treatments and 10 replications, each consisting of a vial containing two nodal segments. After 30 days of cultivation assessed the establishment percentage (development of new leaf primordia), callus formation at the explant base, rooting and number of shoots per explant. The BAP presence in the nutrient medium is not beneficial to the in vitro multiplication of ipe-amarelo tree seedlings. The BAP addition decreases the explants established percentage, rooting, and also the shoots formation, in addition to considerably increase in the calluses formation.

Radiofrequency encoded angular-resolved light scattering

The sensitive, specific, and label-free classification of microscopic cells and organisms is one of the outstanding problems in biology. Today, instruments such as the flow cytometer use a combination of light scatter measurements at two distinct angles to infer the size and internal complexity of cells at rates of more than 10 000/s. However, by examining the entire angular light scattering spectrum, it is possible to classify cells with higher resolution and specificity. Current approaches to performing these angular spectrum measurements all have significant throughput limitations, making them incompatible with other state-of-the-art flow cytometers. Here, we introduce a method for performing complete angular scattering spectrum measurements at high throughput combining techniques from the field of scattering flow-cytometry and radiofrequency communications. Termed Radiofrequency Encoded Angular-resolved Light Scattering, this technique multiplexes angular light scattering in the radiofrequency domain, such that a single photodetector captures the entire scattering spectrum from a particle over approximately 100 discrete incident angles on a single shot basis. As a proof-of-principle experiment, we use this technique to perform scattering measurements over a range of 30° from a tapered optical fiber at a scan rate of 250 kHz.

Use of liquid culture medium and ex vitro rooting for micropropagation of Acacia nilotica (L.) Del. ssp. cupressiformis

A micro-propagation method using liquid culture medium has been developed for Acacia nilotica ssp. cupressiformis. Nodal segments obtained from mature tree (15–20 years old) was used as explants for bud breaking and multiple shoot induction. MS liquid medium with 2.0 mg l−1 of 6-benzylaminopurine (BAP) was the best for shoot bud induction. Further shoots multiplication was achieved by subculturing of in vitro produced shoot clumps (2–3 shoots) in liquid medium supplemented with various concentrations of BAP. Maximum number of shoots (12.57 ± 0.37) and shoot length (10.11 ± 0.42) were recorded in MS liquid medium supplemented with 1.0 mg l−1 of BAP. About 90 % shoots rooted ex-vitro on soilrite within 3–4 weeks when base (3–5 mm) of shoots was treated with 300 mg l−1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the green house and transferred to the polybags. To the best of our knowledge, this is the first report of a micro-propagation protocol for A. nilotica ssp. cupressiformis.

Base to Tip and Long-Distance Transport of Sodium in the Root of Common Reed [Phragmites australis (Cav.) Trin. ex Steud.] at Steady State Under Constant High-Salt Conditions.

We analyzed the directions and rates of translocation of sodium ions (Na(+)) within tissues of a salt-tolerant plant, common reed [Phragmites australis (Cav.) Trin. ex Steud.], and a salt-sensitive plant, rice (Oryza sativa L.), under constant high-salt conditions using radioactive (22)Na tracer and a positron-emitting tracer imaging system (PETIS). First, the test plants were incubated in a nutrient solution containing 50 mM NaCl and a trace level of (22)Na for 24 h (feeding step). Then the original solution was replaced with a fresh solution containing 50 mM NaCl but no (22)Na, in which the test plants remained for >48 h (chase step). Non-invasive dynamic visualization of (22)Na distribution in the test plants was conducted during feeding and chase steps with PETIS. Our results revealed that (22)Na was absorbed in the roots of common reed, but not transported to the upper shoot beyond the shoot base. During the chase step, a basal to distal movement of (22)Na was detected within the root tissue over >5 cm with a velocity of approximately 0.5 cm h(-1). On the other hand, (22)Na that was absorbed in the roots of rice was continuously translocated to and accumulated in the whole shoot. We concluded that the basal roots and the shoot base of common reed have constitutive functions of Na(+) exclusion only in the direction of root tips, even under constant high-salt conditions. This function apparently may contribute to the low Na(+) concentration in the upper shoot and high salt tolerance of common reed.

Psoraleaditurnerae and P.vanberkelae (Psoraleeae, Fabaceae): two new species restricted to the Core Cape Region of South Africa.

Two new species of Psoralea L. are described: Psoralea diturnerae A. Bello, C.H. Stirt. & Muasya, sp. nov. and Psoralea vanberkelae C.H. Stirt., A. Bello & Muasya, sp. nov. Psoralea diturnerae is endemic to the Outeniqua mountains (Camferskloof) and is characterised by a mass of numerous basal shoots out of which emerge 2–3 woody stems up to 2 m tall, 3-foliolate needle-like leaflets at the base of the seasonally growing shoot reducing to one towards the apex and bearing numerous 1–3-flowered axillary inflorescences along its length; each mauve to purple and white flower subtended by a trifid cupulum. Psoralea vanberkelae is characterised by its spreading mounding habit, short tightly packed fleshy leaves, with large impressed papillae, densely glandular short broadly triangular stipules, pale to intense mauve to deep blue flowers, standard with a dark purple central blotch above a M-shaped white patch situated above claw, and khaki seeds with purple flecks.

Distribution of Flavonoids and Cyclohexenyl Chalcone Derivatives in Conventional Propagated andIn Vitro-Derived Field-GrownBoesenbergia rotunda(L.) Mansf.

The distribution patterns of flavonoids and cyclohexenyl chalcone derivatives in conventional propagated (CP) and in vitro-derived (CPA) field-grown plants of an important medicinal ginger, Boesenbergia rotunda, are described. A total of eight compounds were extracted from six organs (rootlet, rhizome, shoot base, maroon stem, stalk, and leaf) of the CP and CPA plants. Five major chromatographic peaks, namely, alpinetin, pinocembrin, pinostrobin, 4-hydroxypanduratin A, and panduratin A, were consistently observed by high performance liquid chromatography. Nonaerial organs had higher levels of flavonoids than the aerial ones for all types of samples. Among the compounds detected, pinostrobin and 4-hydroxypanduratin A were the most abundant flavonoid and cyclohexenyl chalcone derivative, respectively. The distribution and abundance of the bioactive compounds suggested that the shoot base could be more potentially useful for medicinal application than other organs of the plant and may be the site of storage or occurrence of biosynthetic enzymatic activities.

Growth of edible lily (Lilium davidii var. unicolor) and bulb yield responses to potassium fertiliser and plastic film mulching

SummaryEdible lily (Lilium davidii var. unicolor) has economic value due to its food, health products, and gardening uses in China. A continuous 3-year experiment was conducted to determine the effect of potassium (K) supply on plant growth and bulb yield in L. davidii var. unicolor under water-permeable plastic film mulching. Approx. 32,400 seed bulbs (average fresh weight = 21.0 g) were arranged in a completely random design with two patterns of planting (nonmulching or mulching) and three levels of K fertiliser (K0, 0 kg K2O ha-1; K72, 72 kg K2O ha-1; or K144, 144 kg K2O ha-1). It was found that mulching increased seedling heights, basal shoot diameters, leaf numbers, leaf areas, and dry biomass production. L. davidii var. unicolor seedlings also exhibited a strong, but inconsistent response to K supply. An intermediate K supply (K72) increased seedling heights, basal shoot diameters, leaf numbers, leaf areas, and dry biomass production. In contrast, a high K supply (K144) decreased or had little effect on these growth characteristics. Potassium levels, mulching, and their interaction, significantly (P 0.01) decreased the mean length of the longest root. Similarly, marketable bulb yields and bulblet yields were significantly influenced by K level, mulching, and their interaction (P 0.01). Furthermore, the greatest increase in marketable bulb yield occurred between treatments K0 and K72 with or without mulching.

Photochemical Efficiency during the Establishment and Consolidation Phases of in Vitro Pinus radiata Micrograft Made from Scions of Different Ontogenetic Age

The aim of the present study was to evaluate the applicability of maximal photochemical efficiency of photosystem II (Fv/Fm) as an early estimate of P. radiata micrografts viability coming from different position (basal vs. apical) in the ortets. We hypothesize that Fv/Fm variation is a good indicator of micrograft’s viability and phenological stage during micrograft development. The micrografts were established in QL medium supplemented whit 0.1 mg·L-1 IBA and 1 mg·L-1 BAP and cultured at 25°C ± 2°C and 80 μmol photons m-2s-1 of photosynthetic active radiation by 16 h per day. During the establishment and consolidation phase, we found significant differences in Fv/Fm with respect to time and buds positions provenience. During establishment, basal shoot tips have lower Fv/Fm than apical shoot tips, which agrees with the lowest viability (35%). However, during the consolidation phase, the trend changed and basal shoot tips presented higher Fv/Fm than apical shoot tips and showed an increase in ETR and NPQ, with respect to apical shoots and ortet. Although the measurement of fluorescence parameters implies the insertion of the fluorometer sonde in vitro, this implies aseptic considerations, but always conveies a contamination risk. We conclude that fluorescence (Fv/Fm, ETR, NPQ) can be indicators of the micrograft’s development according to the shoot tips position in the ortet and can be useful early-indicators of the scions’ physiological condition during micrograft transition from establishment to consolidation.

In vitro high frequency regeneration through apical shoot proliferation ofHemianthus callitrichoides ‘Cuba’ ‐ a multipurpose ornamental aquatic plant

Hemianthus callitrichoides 'Cuba' is an ornamental aquatic plant used in water gardens and phytoremediation. A protocol for its rapid and reproducible shoot organogenesis from apical shoot explants was developed. Optimum culture conditions for shoot proliferation were tested in MS media containing different concentrations of 6-($\gamma$,$\gamma$-dimethylallylamino)purine (2iP), 6-benzylaminopurine (BAP), 1-phenyl-3-1,2,3-thiadiazol-5-yl urea (TDZ), and gibberellic acid (GA3). Plant growth regulators significantly (P ≤ 0.001) affected the diameter, area, and fresh weight of shoot clumps. On the basis of plant growth, the maximum shoot diameter (3.06 cm), area (6.12 cm), and clump weight (2.85 g) of apical shoots were achieved on MS medium containing 0.50 mg L-1 BAP. Higher concentrations of sucrose (9%) showed visible phenotypic growth inhibition and abnormalities were observed in vitro. The regenerated shoots rooted efficiently on MS medium without growth regulators. The growth pattern of multiple shoots indicated their origin from an enlarged shoot base via the proliferation of apical shoots. All in vitro regenerated plantlets acclimatized in aquariums successfully. The simplicity of the protocol and the direct production of multiple shoots make it a potential system that is highly amenable to true-to-type plant regeneration with the ability to maintain genetic stability.