Search

Buddleia species is commonly used as a perennial for landscaping as a result of its heavy flowers and long bloom period. However, this species has a few concerns such as lack of flower color, excessive growth, and an invasive nature. Attempts to improve Buddleia using conventional breeding methods have resulted in limited success. In this study, mutagenesis by gamma ray irradiation was used to induce genetic variations. In vitro shoot tips of two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were exposed to 0 to 150 Gy gamma rays and then recovered in Murashige and Skoog (MS) medium supplemented with 2.5 μM benzyladenine (BA). Shoots that recovered from the gamma ray treatment were rooted in half-strength MS medium with 0.5 μM naphthalene acetic acid (NAA) and grown in the greenhouse. The growth of shoot tips was inhibited after they were exposed to gamma rays. An average of 50.8% of shoots treated with 50 Gy gamma rays were recovered, whereas only 9.7% and 6.5% of shoots recovered when exposed to 100 and 150 Gy gamma rays, respectively. After transfer to the greenhouse, a few plants showed reduced growth with some dying before they reached the flowering stage. Various variations including characteristics of leaves (shape, size, hairs), stems (shape, internode length, branching), flowers (color, size, and structure), and plant stature were observed. This research demonstrates that in vitro mutation induction using gamma ray irradiation could be a useful protocol to develop new cultivars or genetic materials for further breeding of Buddleia or other related species.

Fabricating theranostic nanoparticles combining multimode disease diagnosis and therapeutic has become an emerging approach for personal nanomedicine. However, the diagnostic capability, biocompatibility, and therapeutic efficiency of theranostic nanoplatforms limit their clinic widespread applications. Targeting to the theme of accurate diagnosis and effective therapy of cancer cells, a multifunctional nanoplatform of aptamer and polyethylene glycol (PEG) conjugated MoS2 nanosheets decorated with Cu1.8S nanoparticles (ATPMC) is developed. The ATPMC nanoplatform accomplishes photoluminescence imaging, photoacoustic imaging, and photothermal imaging for in vitro and in vivo tumor cells imaging diagnosis. Meanwhile, the ATPMC nanoplatform facilitates selective delivery of gene probe to detect intracellular microRNA aberrantly expressed in cancer cells and anticancer drug doxorubicin (DOX) for chemotherapy. Moreover, the synergistic interaction of MoS2 and Cu1.8S renders the ATPMC nanoplatform with superb photothermal conversion efficiency. The ATPMC nanoplatform loaded with DOX displays near-infrared laser-induced programmed chemotherapy and advanced photothermal therapy, and the targeted chemo-photothermal therapy presents excellent antitumor efficiency.

A micropropagation system was developed to clonally propagate juvenile and mature chokecherry plants. Establishment of shoot tips from seedling plants was best on Murashige and Skoog (MS) medium with 5 μM benzyladenine (BA) and 5 μM indolebutyric acid (IBA). Significantly more shoots proliferated on the MS or Driver and Kuniyuki Walnut (DKW) medium than on Woody Plant medium. Rooting of microshoots could be induced on medium with either IBA or naphthaleneacetic acid (NAA). The highest rooting percentage and the greatest number of roots occurred when shoots were placed in a medium with 0.5 or 2.5 μM IBA. More than 20 chokecherry plants shown to have a high level of field tolerance to X-disease were micropropagated for further X-disease resistance testing using the technique described here.

A micropropagation system was developed for mass propagation of ‘Fargo’® (Dakota Pinnacle™), a newly released cultivar of Asian white birch (Betula platyphylla). Shoot tips from the mature, 7-year-old tree were established on 75% strength Murashige and Skoog medium supplemented with 0.1 μM thidiazuron. The greatest shoot proliferation occurred on Woody Plant Medium supplemented with 2.2 μM benzyladenine (BA), solidified with 6.5 g/liter agar, and cultured at 24C (75F). Microshoots were rooted in vitro or ex vitro followed by establishment in the greenhouse. A system to regenerate plantlets from leaves of aseptically cultured shoots was also developed. The optimum conditions for shoot regeneration from leaves included a 2-week dark treatment before exposure to a 16/8 hour light/dark photoperiod, use of large and healthy leaf explants, and culture on the Woody Plant Medium containing 20.0 or 30.0 μM BA. The regenerated shoots proliferated on the micropropagation medium and were divided, and the resulting shoots were rooted ex vitro and acclimated in greenhouse conditions.

Two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were transformed using Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBI121 carrying the neomycin phosphotransferase gene and β-glucuronidase gene (uidA). Transgenic plants were recovered from the Agrobacterium-infected leaf tissues through organogenesis in the selection medium (woody plant medium containing 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 40 mg·L−1 kanamycin). The rate of shoot regeneration from transformed leaf tissues increased from 5.7% to 32% through extending cocultivation time from 3 to 9 days. Integration of marker genes was verified with polymerase chain reaction (PCR) and Southern blot analysis. Southern blot analysis confirmed that one to three copies of transgenes were integrated into the buddleia genome. This transformation system could be used for improvement of buddleia or other related species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime (Cef), carbenicillin (Carb), kanamycin (Km).

Chokecherry (Prunus virginiana L.) was transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pBI121 carrying the neomycin phosphotransferase gene (nptII) and β-glucuronidase (GUS) gene (uidA). Plants were regenerated from the Agrobacterium-infected leaf tissues through organogenesis on woody plant medium (WPM) supplemented with MS (Murashige and Skoog) vitamins, 10 μm 6-benzyladenine (BA), and 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 15 mg·L−1kanamycin (CCK15). Transformation was verified with polymerase chain reaction (PCR) and Southern blot analysis. Four of 150 (2.67%) initial explants produced GUS- and PCR-positive shoots. Southern blot analysis confirmed that the transgenes were integrated into the chokecherry genome. Transgenic in vitro shoots were rooted in half-strength MS medium containing 10 μm naphthalene acetic acid. Rooted plants were transferred to potting mix and grown in the greenhouse. This research shows a potential for future improvement of chokecherry and other Prunus species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime, carbenicillin, kanamycin.

Background Pear (Pyrus spp) is an important fruit species worldwide; however, its genetics and genomic information is limited. Combining the Solexa/Illumina RNA-seq high-throughput sequencing approach (RNA-seq) with Digital Gene Expression (DGE) analysis would be a powerful tool for transcriptomic study. This paper reports the transcriptome profiling analysis of Chinese white pear (P. bretschneideri) using RNA-seq and DGE to better understand the molecular mechanisms in fruit development and maturation of Chinese white pear. Results De novo transcriptome assembly and gene expression analysis of Chinese white pear were performed in an unprecedented depth (5.47 gigabase pairs) using high-throughput Illumina RNA-seq combined with a tag-based Digital Gene Expression (DGE) system. Approximately, 60.77 million reads were sequenced, trimmed, and assembled into 90,227 unigenes. These unigenes comprised 17,619 contigs and 72,608 singletons with an average length of 508 bp and had an N50 of 635 bp. Sequence similarity analyses against six public databases (Uniprot, NR, and COGs at NCBI, Pfam, InterPro, and KEGG) found that 61,636 unigenes can be annotated with gene descriptions, conserved protein domains, or gene ontology terms. By BLASTing all 61,636 unigenes in KEGG, a total of 31,215 unigenes were annotated into 121 known metabolic or signaling pathways in which a few primary, intermediate, and secondary metabolic pathways are directly related to pear fruit quality. DGE libraries were constructed for each of the five fruit developmental stages. Variations in gene expression among all developmental stages of pear fruit were significantly different in a large amount of unigenes. Conclusion Extensive transcriptome and DGE profiling data at five fruit developmental stages of Chinese white pear have been obtained from a deep sequencing, which provides comprehensive gene expression information at the transcriptional level. This could facilitate understanding of the molecular mechanisms in fruit development and maturation. Such a database can also be used as a public information platform for research on molecular biology and functional genomics in pear and other related species.

Cadmium (Cd) is a widespread soil pollutant and poses a significant threat to human health via the food chain. Large phenotypic variations in Cd concentration of radish roots and shoots have been observed. However, the genetic and molecular mechanisms of Cd accumulation in radish remain to be elucidated. In this study, a genetic linkage map was constructed using an F(2) mapping population derived from a cross between a high Cd-accumulating cultivar NAU-Dysx and a low Cd-accumulating cultivar NAU-Yh. The linkage map consisted of 523 SRAP, RAPD, SSR, ISSR, RAMP, and RGA markers and had a total length of 1,678.2 cM with a mean distance of 3.4 cM between two markers. All mapped markers distributed on nine linkage groups (LGs) having sizes between 134.7 and 236.8 cM. Four quantitative trait loci (QTLs) for root Cd accumulation were mapped on LGs 1, 4, 6, and 9, which accounted for 9.86 to 48.64 % of all phenotypic variance. Two QTLs associated with shoot Cd accumulation were detected on LG1 and 3, which accounted for 17.08 and 29.53 % of phenotypic variance, respectively. A major-effect QTL, qRCd9 (QTL for root Cd accumulation on LG9), was identified on LG 9 flanked by NAUrp011_754 and EM5me6_286 markers with a high LOD value of 23.6, which accounted for 48.64 % of the total phenotypic variance in Cd accumulation of F(2) lines. The results indicated that qRCd9 is a novel QTL responsible for controlling root Cd accumulation in radish, and the identification of specific molecular markers tightly linked to the major QTL could be further applied for marker-assisted selection (MAS) in low-Cd content radish breeding program.

Periwinkle, a perennial commonly used as a summer bedding plant, is known as the source of vinca alkaloids used to treat lymphocytic leukemia and Hodgkin's disease. It is also one of the natural hosts of many phytoplasma diseases, such as X-disease of major Prunus species, aster yellows, and ash yellows diseases. Therefore, periwinkle is an ideal plant species for phytoplasma disease research, such as disease transmission, species resistance, and resistant gene screening. Periwinkle tissue culture was established by incubating sterile seeds in hormone-free Murashige and Skoog (MS) medium. Plants were successfully regenerated from in vitro leaf tissues of periwinkle. Adventitious shoots were induced when leaf tissues were cultured on Murashige and Skoog (MS) medium or woody plant medium (WPM) supplemented with benzyladenine (BA) and naphthaleneacetic acid (NAA). Nearly 75% of leaf explants produced shoots in both media with 10–20 μm BA and 1 μm NAA. A mean of 4.3 shoots was produced from each explant cultured on WPM, whereas only 2 shoots were produced on MS medium under 16-h photoperiod. Leaf explants under dark treatment for 2 weeks produced big callus only, indicating that light is necessary for shoot formation. Most adventitious shoots were induced from the joint of leaf blade and petiole. In vitro shoots (>1.5 cm) were easily rooted in half-strength MS medium. Addition of NAA dramatically increased root number, with more than 20 roots being induced in 5 μm NAA medium. Rooted plants were transferred to potting medium and grown in a greenhouse.

Many woody plants, including some birch species, can be cloned using such in vitro techniques as pre-existing meristem culture, organogenesis, and embryogenesis. However, clonal fidelity of in vitro-derived plants is always a big concern because somaclonal variations may be induced during the entire in vitro process. To address this issue, we used random amplified polymorphic DNA (RAPD) markers to determine the genetic stability of in vitro-propagated plants of Betula platyphylla `Fargo'. Forty-two greenhouse-grown birch plants derived from a 10-year shoot tip culture (shoot-derived) and 42 in vitro plants regenerated from leaf tissues (regenerated) were randomly selected and evaluated for their genetic fidelity by RAPD. To date, 20 primers (C1-C20, Operon Technologies) were screened for all 84 plants. Only strong bands that are conservative were scored. Each primer generated a unique set of amplification products. Most of scoreable bands are ranged from 350 to 1800 bp. A total of 3696 fragments were amplified from 42 shoot-derived plants by all 20 primers with an average of 4.4 bands per primer, in which 6 primers produced polymorphic bands, indicating some genetic variations within shoot-derived plants. Nineteen out of 20 primers yielded 2772 clear and reproducible bands (an average of 3.47 per primer) from 42 regenerated plants with no significant variations being detected. Our preliminary results showed that in vitro regenerated plants are genetically uniform. However, a long-term tissue culture might result in a few genetic variations of birch species.

In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.

An Agrobacterium-mediated transformation system was developed for chokecherry (Prunus virginiana L.), one of the most popular native small tree or large shrub species for resource conservation and wildlife habitat in North America. Leaf tissues from in vitro plants previously maintained in MS medium with 2.5 μm BA were co-cultivated on woody plant medium (WPM) containing 10 μm BA and 200 μm acetosyringone with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uid A gene encoding for β-glucuronidase (GUS) and the npt II gene encoding neomycin phosphotransferase II. Infected leaf explants were disinfected in sterile water and antibiotics and then transferred to WPM containing 10 μM BA and the antibiotics cefotaxime, carbenicillin, and kanamycin (CCK) for shoot regeneration at 25 °C with a 16-hour photoperiod. Agrobacterium concentration, pre-conditioning of explants, application of acetosyringone, infection time, and kanamycin tolerance of leaf tissues were evaluated for effects on transformation efficiency. Regeneration of chokecherry shoots on kanamycin-containing medium and screening by GUS histochemical assays showed that both the npt II and the uid A genes were successfully transferred into chokecherry. The transformation will be further confirmed by polymerase chain reaction (PCR) and Southern blot analyses.

A high-efficiency regeneration/transformation system was developed for three elite aspen hybrids (Populus tremuloides × P.tremula, P.tremuloides × P.davidiana, and P. × canescens × P. grandidentata). On both modified MS medium for aspen (MSA) and Woody Plant Medium (WPM) supplemented with zeatin (2.0 mg/L) and NAA (1.0 mg/L), nearly 100% leaf explants formed calli, of which 80% to 100% regenerated into shoots on both media with 2.0mg/L zeatin and 0.01 mg/L TDZ. Bacterial concentration, pH value of the co-cultivation medium, and acetosyringone were evaluated for enhancing transformation efficiency. Agrobacterial concentration at 1.0 Absorbance at OD600 was better than at 0.1, 0.5 Abs, yielding 80% and 75% of callus induction rates from agrobacterium harboring CaMV35s and Heat shock promoter constructs, respectively. The pH of co-cultivation medium, ranging 5.0 to 5.9, did not have any effect on transformation frequency. Acetosyringone was added to the co-cultivation medium and/or to the callus induction medium, the induction of kanamycin-resistant callus increased from 70% to 80% to 90% to 100%, and the size of callus also increased. Acetosyringone had no effect on shoot regeneration from kanamycin-resistant calli. Regenerated aspen shoots were screened on the kanamycin-containing medium, and confirmed by GUS histochemical assay. The GUS-positive plants were further confirmed by polymerase chain reaction, showing that the nptII, uidA, and rolB genes were integrated into the aspen genomes.

Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.

`Xiao Fang Shi' is a rare, dwarf cultivar of persimmon (Diospyros kaki Linn cv.) recently found north of Shanghai, China. The tree starts to bear fruit at 2 years of age, while standard trees start fruiting at 5 or 6 years of age. Dwarf and standard cultivars have about equal spring shoot growth, but the dwarf cultivar has little fall growth. To determine the mechanisms of dwarfness and early fruiting, enzyme-linked immunosorbant assay (ELISA) was used to analyze the endogenous indoleacetic acid (IAA), gibberellic acid (GA1+3) and abscicic acid (ABA) contents in leaves and shoot tips of dwarf (`Xiao Fang Shi') and standard (`Da Fang Shi' and `Zhu Sha Hong') persimmon. The measurement was done during the entire growing season. The results showed that IAA, GA1+3, and ABA contents were influenced by cultivars, ages of trees, and phenophases. The dwarf cultivar `Xiao Fang Shi' has lower IAA and GA1+3 but higher ABA contents than the two standard cultivars during the growing season. These correlations are especially evident when the fruit is ripening. The correlation coefficiency between contents of IAA and GA1+3 and tree height was 0.9704, while that between ABA content and tree height is –0.9697. The low IAA and GA, and high ABA contents may be responsible for little shoot growth of the dwarf cultivar in the fall.