Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1) protein but not its iron(II) transport function.

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1-2, compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short-term 55 Fe uptake rates were uninformative concerning IRT1-mediated transport. Overall irt1-like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis of irt1-2, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops.

Cryopreservation of Plant Shoot Tips of Potato, Mint, Garlic, and Shallot Using Plant Vitrification Solution 3.

Cryopreservation of shoot tips facilitates long-term storage of plant genetic resources which can otherwise only be propagated vegetatively. The vitrification approach using the cryoprotectant plant vitrification solution 3 (PVS3, 50% sucrose and 50% glycerol) is easy to handle, has shown to produce high regrowth percentages in a number of potato, mint, garlic, and shallot accessions, and is, thus, highly suitable for routine cryopreservation of plant genetic resources. In the current chapter, the vitrification procedure is described for potato, mint, garlic, and shallot and includes details about modifications for the different plant species. Special emphasis is given on the preparation of the different culture media, solutions, the culture conditions prior and post-cryopreservation, and the preparation of the shoot tips from different sources. Furthermore, protocols to introduce plants into in vitro culture and methods to estimate cryopreservation success are provided.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

In Vitro Micropropagation of Industrially and Medicinally Useful Plant Aloe trichosantha Berger Using Offshoot Cuttings.

This study was aimed to develop in vitro micropropagation protocol of Aloe trichosantha Berger using offshoots as explants. MS media supplemented with plant growth regulators helped explants develop shoots within about 14 to 17 days. The mean number of days to shooting has decreased from 16.8 ± 0.8 with 0.5/0.5 mg/L BAP/NAA supplement to 15.5 ± 0.5 with 2.0/0.5 mg/L BAP/NAA. While the mean shoot number has increased with increasing the concentration of BAP supplements, the reverse was true with mean shoot lengths, whereas supplement of 2.0/0.5 mg/L BAP/NAA has generated significantly more shoots (17 ± 3.8), and longer shoots were produced with the addition of 0.5/0.5 and 1.0/0.5 mg/L BAP/NAA. In regard to rooting, though higher concentrations of NAA have resulted in quick rooting, the rooting performance in terms of mean number and length of roots was better with low concentrations. All the plantlets subjected to greenhouse acclimatization in cocopeat have survived. Secondary acclimatization in composted and manured soil media has also resulted in 93 to 95% survival rate. Lighting conditions (nursery shade or direct sunlight) of secondary acclimatization did not lead to any difference in the survival rate of the plantlets.

Endogenous auxin determines the pattern of adventitious shoot formation on internodal segments of ipecac.

MAIN CONCLUSION: Endogenous auxin determines the pattern of adventitious shoot formation. Auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots. Adventitious shoot formation is required for the propagation of economically important crops and for the regeneration of transgenic plants. In most plant species, phytohormones are added to culture medium to induce adventitious shoots. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Thus, ipecac culture allows us to investigate the effects of endogenous phytohormones during adventitious shoot formation. In phytohormone-free culture, adventitious shoots were formed on the apical region of the internodal segments, and a high concentration of IAA was detected in the basal region. To explore the relationship between endogenous auxin and adventitious shoot formation, we evaluated the effects of auxin transport inhibitors, auxin antagonists, and auxin biosynthesis inhibitors on adventitious shoot formation in ipecac. Auxin antagonists and biosynthesis inhibitors strongly suppressed adventitious shoot formation, which was restored by exogenously applied auxin. Auxin biosynthesis and transport inhibitors significantly decreased the IAA level in the basal region and shifted the positions of adventitious shoot formation from the apical region to the middle region of the segments. These data indicate that auxin determines the positions of the shoots formed on internodal segments of ipecac. Only one of the shoots formed grew vigorously; this phenomenon is similar to apical dominance. When the largest shoot was cut off, other shoots started to grow. Naphthalene-1-acetic acid treatment of the cut surface suppressed shoot growth, indicating that auxin produced in the dominant shoot is transported to the internodal segment and suppresses growth of other shoots.

Combining Thermotherapy with Cryotherapy for Efficient Eradication of Apple stem grooving virus from Infected In-vitro-cultured Apple Shoots.

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Rice callus suspension culture inhibits growth of cell lines of multiple cancer types and induces apoptosis in lung cancer cell line.

BACKGROUND: Cancer is one of the leading cause of mortality. Even though efficient drugs are being produced to treat cancer, conventional medicines are costly and have adverse effects. As a result, alternative treatments are being tried due to their low cost and little or no adverse effects. Our previous study identified one such alternative in rice callus suspension culture (RCSC) which was more efficient than Taxol® and Etoposide, in reducing the viability of human colon and renal cancer cells in culture with minimal or no effect on a normal cell line. METHODS: In this study, we tested the effect of RCSC by studying the dynamics of lactate dehydrogenase (LDH) in lung cancer cell lines (NCI-H460 and A549), breast cancer cell lines (MDA-MB-231 and MCF-7) and colorectal cancer cell lines (SW620 and Caco-2) as well as their normal-prototypes. Complementary analysis for evaluating membrane integrity was performed by estimating LDH release in non-lysed cells and cell viability with WST-1 assay. Fluorescence microscopy with stains targeting nucleus and cell membrane as well as caspase 3/7 and Annexin V assays were performed. Real-time quantitative RT-PCR was performed to evaluate expression of 92 genes associated with molecular mechanisms of cancer in RCSC treated ling cancer cell line, NCI-H460 and its normal prototype, MRC-5. High performance liquid chromatography (HPLC) was used to collect RCSC fractions, which were evaluated on NCI-H460 for their anti-cancer activity. RESULTS: Lower dilutions of RCSC showed maximum reduction in total LDH indicating reduced viability in majority of the cancer cell lines tested with minimal or no effect on normal cell lines compared to the control. Complementary analysis based on LDH release in non-lysed cells and WST-1 assay mostly supported total LDH results. RCSC showed the best effect on the lung non-small carcinoma cell line, NCI-H460. Fluorescence microscopy analyses suggested apoptosis as the most likely event in NCI-H460 treated with RCSC. Gene expression analysis identified significant upregulation of cJUN, NF-κB2 and ITGA2B in NCI-H460 which resulted most likely in the arrest of cell cycle progression and induction of apoptotic process. Further, HPLC-derived RCSC fractions were less effective in reducing cell viability than whole RCSC suggesting that a holistic approach of using RCSC is a better approach in inhibiting cancer cell proliferation. CONCLUSIONS: RCSC was found to be an effective anti-cancer agent on cell lines of multiple cancer types with the best effect on lung cancer cell lines. A possible mechanism for the anticancer activity of RCSC is through induction of apoptosis as observed in the lung cancer cell line, NCI-H460.

Characterization of the AtSPX3 Promoter Elucidates its Complex Regulation in Response to Phosphorus Deficiency.

AtSPX3, responding to phosphate (Pi) deficiency by its expression, is an important gene involved in Pi homeostasis in Arabidopsis. To understand its transcriptional regulation, we characterized the AtSPX3 promoter by distal truncation, internal deletion and mutation of the predicted cis-elements, and identified multiple cis-elements responsive to Pi status. The P1BS (AtPHR-binding site) and AtMyb4 (putative MYB4-binding site) elements were two main cis-elements in the AtSPX3 promoter. P1BS is essential and has a dosage effect for activating expression of the gene under Pi deficiency, while the element AtMyb4 possesses a dual function: one is to enhance AtSPX3 expression in roots under Pi deficiency, and the other one is to repress AtSPX3 expression in shoots under both Pi deficiency and sufficiency. Moreover, we confirmed that AtPHR1, a key transcription factor in Pi homeostasis of plants, was required for the negative regulation function of the AtMyb4 element in shoots. Additionally, we also found that the AtSPX3 promoter had a length limitation for activating gene expression. Generally, our findings in this work are useful for understanding the molecular regulation mechanism of genes involved in Pi uptake and homeostasis.

Methyl jasmonate, yeast extract and sucrose stimulate phenolic acids accumulation in Eryngium planum L. shoot cultures.

Eryngium planum L. has been reported as a medicinal plant used in traditional medicine in Europe. The tissue cultures may be an alternative source of the biomass rich in desired bioactive compounds. The purpose of this study was to investigate the influence of the biotechnological techniques on the selected phenolic acids accumulation in the agitated shoot cultures of E. planum. Qualitative and quantitative analyses of those compounds in 50% aqueous - methanolic extracts from the biomass were conducted by applying the HPLC method. Methyl jasmonate (MeJA), yeast extract (YE) and sucrose (Suc) stimulated accumulation of the phenolic acids: rosmarinic (RA), chlorogenic (CGA) and caffeic (CA) in in vitro shoot cultures. Cultivation of shoots in liquid MS media supplemented with 1.0 mg L(-1) 6-benzyladenine and 0.1 mg L(-1) indole-3-acetic acid in the presence of 100 µM MeJA for 48h was an optimum condition of elicitation and resulted in approximately 4.5-fold increased content of RA + CGA + CA in plant material compared to the control (19.795 mg g(-1) DW, 4.36 mg g(-1) DW, respectively). The results provide the first evidence that the selected phenolic acids can be synthesized in elicited shoot cultures of flat sea holly in higher amount than in untreated shoots.

The effect of selenium on the subcellular distribution of antimony to regulate the toxicity of antimony in paddy rice.

Selenium (Se) can alleviate the toxicity of antimony (Sb) in plants; however, the associated mechanisms have not been fully clarified. In this study, we hypothesize that Se can affect the subcellular distribution of Sb to regulate Sb toxicity. To test our hypothesis, two nested hydroponic experiments were performed by using paddy rice (Fengmeizhan). The results showed that Sb exerted toxic effects on the growth of paddy rice, and Se caused beneficial effects that were limited to the shoot growth. In general, Se and Sb mutually showed antagonistic effects on their uptake and concentrations in different subcellular fractions. However, in some cases, the stimulation effects of Sb on the Se concentration in chlorophyll (Chl) and cytosol (Cy) fractions or of Se on the Sb concentration in the cell wall fraction (Cw) were also observed in the shoots, which might suggest that Sb detoxification by Se is also related to the migration of both Se and Sb in cells. Selenium and Sb were primarily concentrated in the Cw and Cy, suggesting the important roles of these two fractions in detoxifying Se and Sb. When paddy rice was subjected to increasing Sb concentrations and a fixed Se concentration, most of the Se in the shoots was sequestered in the Cy (59.81-79.51% of total Se) and more Se was transferred into the inner cell from Cw; however, in the roots, Se was primarily concentrated in the Cw (53.28-72.10%). When paddy rice was exposed to increasing Se concentrations with a fixed Sb concentration, the Cw in both the shoots and roots might play an important role in binding Se, especially in the roots where up to 78.92% of the total Se was sequestered in the Cw.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Phytoremediation of heavy and transition metals aided by legume-rhizobia symbiosis.

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

The influence of L-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. ex Regel shoot culture.

Phenolic acids are an important group of plant secondary metabolites with various, valuable therapeutic properties. Apart from plants growing in the open air, tissue cultures can be an alternative source of the secondary metabolites. The yield of their accumulation in in vitro cultures can be increased by different methods, including culture medium supplementation with precursors, elicitors and changing the standard amounts of the medium components. The purpose of this study was to investigate the influence of the precursor (L-phenylalanine), the elicitor (methyl jasmonate) and a higher sucrose concentration on the phenolic acids accumulation in the agitated shoot cultures of Exacum affine Balf. f. ex Regel (Gentianaceae). Qualitative and quantitative analyses of the phenolic acids in methanolic extracts from the biomass were conducted by applying the HPLC method. Fourteen phenolic acids and cinnamic acid were found in all samples. The total content of free phenolic acids increased from approximately 0.242% to 0.635% (2.6-fold) and the total content of the whole phenolic acids (free and bound) - from 0.712% to 1.160% (1.6-fold). The studies show that the best variant for the accumulation of most of the identified phenolic acids contained 6% of sucrose (double the standard amount), L-phenylalanine 1.6 gL(-1) of medium and methyl jasmonate 100 µM. The analysis of the results in the experiment presented here showed that it is possible to increase the accumulation of the phenolic acids in Exacum affine shoot cultures - by adding the precursor (L-phenylalanine), the elicitor (methyl jasmonate) and by increasing the sucrose concentration.

Analysis of selenium accumulation, speciation and tolerance of potential selenium hyperaccumulator Symphyotrichum ericoides.

Symphyotrichum ericoides was shown earlier to contain hyperaccumulator levels of selenium (Se) in the field (>1000 mg kg(-1) dry weight (DW)), but only when growing next to other Se hyperaccumulators. It was also twofold larger next to hyperaccumulators and suffered less herbivory. This raised two questions: whether S. ericoides is capable of hyperaccumulation without neighbor assistance, and whether its Se-derived benefit is merely ecological or also physiological. Here, in a comparative greenhouse study, Se accumulation and tolerance of S. ericoides were analyzed in parallel with hyperaccumulator Astragalus bisulcatus, Se accumulator Brassica juncea and related Asteraceae Machaeranthera tanacetifolia. Symphyotrichum ericoides and M. tanacetifolia accumulated Se up to 3000 and 1500 mg Se kg(-1) DW, respectively. They were completely tolerant to these Se levels and even grew 1.5- to 2.5-fold larger with Se. Symphyotrichum ericoides showed very high leaf Se/sulfur (S) and shoot/root Se concentration ratios, similar to A. bisulcatus and higher than M. tanacetifolia and B. juncea. Se X-ray absorption near-edge structure spectroscopy showed that S. ericoides accumulated Se predominantly (86%) as C-Se-C compounds indistinguishable from methyl-selenocysteine, which may explain its Se tolerance. Machaeranthera tanacetifolia accumulated 55% of its Se as C-Se-C compounds; the remainder was inorganic Se. Thus, in this greenhouse study S. ericoides displayed all of the characteristics of a hyperaccumulator. The larger size of S. ericoides when growing next to hyperaccumulators may be explained by a physiological benefit, in addition to the ecological benefit demonstrated earlier.

Localization of the Bacillus subtilis beta-propeller phytase transcripts in nodulated roots of Phaseolus vulgaris supplied with phytate.

Soil organic phosphorus (Po) such as phytate, which comprises up to 80 % of total Po, must be hydrolyzed by specific enzymes called phytases to be used by plants. In contrast to plants, bacteria, such as Bacillus subtilis, have the ability to use phytate as the sole source of P due to the excretion of a beta-propeller phytase (BPP). In order to assess whether the B. subtilis BPP could make P available from phytate for the benefit of a nodulated legume, the P-sensitive recombinant inbred line RIL147 of Phaseolus vulgaris was grown under hydroaeroponic conditions with either 12.5 µM phytate (C6H18O24P6) or 75 µmol Pi (K2HPO4), and inoculated with Rhizobium tropici CIAT899 alone, or co-inoculated with both B. subtilis DSM 10 and CIAT899. The in situ RT-PCR of BPP genes displayed the most intense fluorescent BPP signal on root tips. Some BPP signal was found inside the root cortex and the endorhizosphere of the root tip, suggesting endophytic bacteria expressing BPP. However, the co-inoculation with B. subtilis was associated with a decrease in plant P content, nodulation and the subsequent plant growth. Such a competitive effect of B. subtilis on P acquisition from phytate in symbiotic nitrogen fixation might be circumvented if the rate of inoculation were reasoned in order to avoid the inhibition of nodulation by excess B. subtilis proliferation. It is concluded that B. subtilis BPP gene is expressed in P. vulgaris rhizosphere.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

In vitro adventitious shoot regeneration via indirect organogenesis from inflorescence explants and peroxidase involvement in morphogenesis of Populus euphratica Olivier.

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0 mg L(-1) 6-benzylaminopurine (BA) and 0.5 mg L(-1) α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0 mg L(-1) indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5 mg L(-1) IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8 days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.

'Personalisation' of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects.

Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5 percent glycerol + 17.5 percent sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90 percent (37.5 percent glycerol + 15% DMSO + 15 percent EG + 22.5 percent sucrose) for 10 to 30 min or B1-100 percent (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40 degree C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.

Phenolic compound production in relation to differentiation in cell and tissue cultures of Larrea divaricata (Cav.).

The lignan nordihydroguaiaretic acid (NDGA) and its derivatives existing in Larrea divaricata species show a wide range of pharmacological activities which makes this genus an interesting target to consider the plant in vitro cultivation systems as a feasible alternative source for their production. These compounds are potentially useful in treating diseases related to heart condition, asthma, arteriosclerosis, viral and bacterial infections, inflammation and cancer. In the present study, calli, cell suspension cultures, and in vitro and wild plants of L. divaricata were investigated for their potential to synthesize phenolic compounds. Calli, both with and without organogenesis, produced NDGA and quercetin, as did plantlet and wild plants. NDGA was also produced by the cell suspension cultures, together with p-coumaric acid, ferulic acid and sinapyl alcohol. The capacity of undifferentiated tissues to form phenolic compounds is very limited, but when the calli underwent organogenesis, developing mainly adventitious shoots, the phenolic compound production increased significantly. Plantlets regenerated from adventitious shoots of L. divaricata calli did not show the same phenolic pattern as wild plants, with levels of NDGA and quercetin being 3.6- and 5.9-fold lower, respectively.