Efficient,reproducible <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum using heat treatment of immature embryos

A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal 43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at 43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2 to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is several-fold higher than published frequencies.     

High-frequency vibration improve callus growth via antioxidant enzymes induction in <Emphasis Type="Italic">Hyoscyamus kurdicus</Emphasis>

Effect of high-frequency vibration on growth rate, membrane stability and activities of some antioxidant enzymes were studied in callus tissues of Hyoscyamus kurdicus. Calli initiated from leaf (LE), shoot (SE) and root (RE) explants, and sinusoidal vibrations at 0, 50, 100 and 150 Hz for 30 min were applied on the H. kurdicus calli. Results showed that sinusoidal vibration at 50 and 100 Hz promoted the growth rate as compared to control, and the optimum growth was found at 50 Hz. Sinusoidal vibration increased significantly protein and proline contents and activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POX) enzymes, and decreased total carbohydrate, H₂O₂ level and CAT activity as compared to control. Lipid peroxidation also decreased under sinusoidal vibration in all the calli, and the maximum percentage of decrease was observed at 50 Hz. Native polyacrylamide gel electrophoresis indicated different isoform profiles in vibration treated and untreated plants concerning antioxidant enzymes. The responses of different types of calluses were different, and RE callus showed the highest growth, membrane stability and antioxidant enzymes activity as compared to LE and SE calli. These results suggest sinusoidal vibration at optimum frequency could improve callus growth by induction of antioxidative enzymes activity and membrane stability in calli of H. kurdicus.     

Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase,key genes involved in camptothecin biosynthesis in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne (Nyssaceae)

Background  

Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Camptothecin accumulation in various organ cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g⁻¹ DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g⁻¹ DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g⁻¹ DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g⁻¹ FW were determined at 4 and 8 immersion cycles daily (IC d⁻¹), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Dynamic analysis of olive trees in intensive orchards under forced vibration

The mechanical harvesting of fruit for oil production in an intensive olive tree orchard is generally accomplished by applying vibration to the tree’s trunk. This vibration is consequently transmitted to the branches, causing the fruit to detach. Although this practice is commonly used, the effects on tree behavior under forced vibration are not firmly established. Dynamic analysis was performed on 17 olive trees (Olea europaea L.) growing in an intensively-managed orchard using modal testing techniques. Modal parameter identification was focused inside the range excitation frequency used by the most commonly available trunk shakers on the market. The olive trees featuring a low morphological variability and modal parameters were obtained for a representative olive tree. The first two modes of vibration of the main tree frame were identified with damping ratios of 26.9 and 17.1% and natural frequencies of 20.2 and 37.7 Hz, respectively. A third mode of vibration of less importance was found at a higher frequency. Therefore, many local modes of vibration were detected near these natural frequencies, primarily located on secondary branches. During the testing, the olive trees behaved like a damped harmonic oscillator with predominantly mass damping in these modes.     

Dependence of UV-B-induced camptothecin production on nitrate reductase-mediated nitric oxide signaling in Camptotheca acuminata suspension cell cultures

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.     

喜树各器官解剖学及其喜树碱含量测定的研究

运用植物解剖学方法研究了喜树各器官的结构,对喜树不同器官中喜树碱含量进行了HPLC法测定。结果表明:(1)喜树根尖纵切面的结构由根冠、分生区、伸长区和根毛区4个部分组成。根和茎的初生结构都由表皮、皮层和维管柱3部分组成;在根和茎初生结构的皮层和髓部具有被锇酸染成黑色的嗜锇细胞;在次生生长中,根的木栓形成层起源于中柱鞘,茎的木栓形成层起源于皮层细胞;随着次生维管组织的增加,茎的中央形成髓腔。(2)叶片由表皮、叶肉和叶脉组成,叶肉细胞中分布有嗜锇细胞;光学显微镜和扫描电子显微镜观察显示,喜树叶的上表皮没有气孔器分布;下表皮气孔器的保卫细胞呈肾形,没有副卫细胞,被几个表皮细胞不规则的围绕,属于无规则型;在上下表皮均分布着单细胞非腺毛和2种类型的腺毛,其中下表皮的分布相对稠密;上表皮腺毛呈球形,下表皮腺毛大部分为长卵形,其中间杂着一些球形腺毛;观察发现,越幼嫩的叶中,分布的腺毛和非腺毛越多。(3)喜树不同器官中喜树碱含量以幼叶和种子中较高,木质部中较低,髓中含量最少;随着叶的发育成熟,叶中喜树碱的含量逐渐降低,且同一叶的不同部位,喜树碱含量也有差异;喜树幼苗发育过程中喜树碱含量也在不断变化。     

Effects of light on production of camptothecin and expression of key enzyme genes in seedlings of <Emphasis Type="Italic">Camptotheca acuminate</Emphasis> Decne

Camptotheca acuminata (C. acuminata) is utilized in preparation of drugs and as constituent in functional foods of China due to high camptothecin (CPT) content in different plant parts. Light intensity is one of the most critical factors which affect plant growth and secondary metabolites. Pot experiment was conducted to study the effect of light intensity (i.e., 100 % irradiance (control), 75 % irradiance, 50 % irradiance and 25 % irradiance) on contents of CPT, activity of enzymes and genes expression related to CPT biosynthesis of C. acuminata seedlings. The study examined total leaf biomass, CPT content, activities of tryptophan synthase (TSB) and tryptophan decarboxylase (TDC), and relative expression of TSB, TDC1, and TDC2 genes. Plants grown in 75 % irradiance possessed the greatest leaf biomass compared with 100 % light irradiance. Highest values of CPT contents were found after 60 days in plants grown in 50 % irradiance, followed by 25, 75 % and full sunlight. Furthermore, activities of TSB, TDC and relative expression of genes of TSB, TDC1, and TDC2, were significantly increased after 60 days of 50 % irradiance compared with full sunlight. Irradiance of 50 % up-regulated the expression of CPT biosynthesis-related genes and induced CPT biosynthesis. In addition to that lower or higher irradiance inhibited the expression of CPT biosynthesis-related genes and CPT biosynthesis. It is concluded that manipulating light intensity can be an effective means to achieve highest CPT yield in medicinal plants.     

Light-regulated betacyanin accumulation in euhalophyte <Emphasis Type="Italic">Suaeda salsa</Emphasis> calli

Suaeda salsa calli cultured in darkness for 28 days were transferred to Murashige and Skoog (MS) media containing 0.5 mg l⁻¹ Thidiazuron (TDZ) and 1.0 mg l⁻¹ naphthaleneacetic acid (NAA) under different light conditions for 10 days to investigate effect of light on betacyanin accumulation and its relation to activity and expression of tyrosinase and expression of dopa-4,5-dioxygenase gene (DODA). Both light quality and quantity affected betacyanin synthesis of S. salsa calli. 80 μmol m⁻² s⁻¹ of white light intensity was optimal for cell growth and betacyanin accumulation of S. salsa calli. DODA mRNA levels and tyrosinase activity were consistent with the response of betacyanin content to different light quality. However, the protein levels of tyrosinase extracted from S. salsa calli response to different light quality were constant. These results suggest that betacyanin metabolism in S. salsa calli is regulated by different light quality through the regulation of genes such as DODA and tyrosinase activity but not via alteration of the protein amount of tyrosinase.     

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g^??1 dry wt (up to 11 mg g^??1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.

     

Trichomes in Camptotheca acuminata Decaisne (Nyssaceae): Morphology,distribution, structure,and secretion

Camptotheca acuminata is a main source of the anti-cancer drug camptothecin (CPT). In this species, several studies have observed non-glandular trichomes (NGTs) and glandular trichomes (GTs). It has been assumed that GTs contain CPT, yet this has not been proven and no information is available on the accumulation of other secondary metabolites. The objective of this study was to describe the morphology, distribution and structure of C. acuminata trichomes and to investigate the chemical nature of the substances secreted by GTs. Light and fluorescence microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to determine the morphology, distribution and structure of GTs and NGTs. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses were carried out to confirm the presence of CPT in GTs, and histochemical tests were performed to investigate the presence of other secondary metabolites. C. acuminata possesses two types of GTs (GT1 and GT2), which differ in terms of their morphology, pattern of distribution and accumulated substances. The chemical analyses demonstrated that both GT1 and GT2 accumulate CPT. Histochemical analysis showed that phenols accumulate in the vacuole of GT2s. No isoprenoids were detected in GTs.     

Frequency response of Pinus Pinea L. for selective cone harvesting by vibration

The stone pine (Pinus pinea L.) is exploited for wood production and its edible kernels. Cones take 3 years to mature, while other newer cones are growing on the tree. Currently, mechanical cone harvesting by tree vibration drives the profitability and development of this crop in forest ecosystems. However, the adaptation of vibration parameters is necessary to avoid damage to the tree and for the implementation of good harvesting practices. Direct measurements of acceleration transmissibility along fruit-bearing branches under controlled laboratory conditions showed that vibrations in the frequency range of 18.0 ± 5.3 Hz were capable of producing resonance phenomena in mature cones. Morphological changes produced in the mature cones, especially in the stalk and total weight, amplified the acceleration transmissibility, providing more favourable conditions for fruit detachment. Field tests with stone pines and a trunk shaker confirmed the potential of selective mechanical harvesting, which is conditional on the vibration parameters applied. The frequency range of the resonance of the mature cone proved to be suitable for vibration of the tree at its trunk. The quality and efficiency of the mechanical harvesting were determined to be inversely related parameters during harvesting by vibration. Trunk vibration at a frequency of 18 Hz and approximate acceleration of 65 m/s² achieved a high harvest quality, with limited detachment of shoots and unripe cones, and a harvesting efficiency near 85%.     

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

Camptothecin and 10-hydroxycamptothecin from<Emphasis Type="Italic"> Camptotheca acuminata</Emphasis> hairy roots

Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae. Bark and seeds are currently used as sources for the drug. Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach. Cultures of differentiated cell types may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000. Integration of the genes responsible for the hairy-root phenotype (rol genes) into the plant genome was verified by DNA gel blot analysis. The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium. Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.Abbreviations CPT Camptothecin - DR Dry weight - FW Fresh weight - HCPT 10-Hydroxycamptothecin - HPLC High-performance liquid chromatography - kb Kilobases - NAA 1-Napththalenacetic acid - TLC Thin-layer chromatographyCommunicated by K.K. Kamo     

An efficient regeneration system and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese upland rice cultivar Handao297

A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants. Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots by five to tenfold in comparison to medium without TDZ.     

Freezing of dehydrated calli of spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) in liquid nitrogen and their morphogenetic potential

A cryopreservation method developed earlier was modified for freezing of calli derived from mature embryos of four spring wheat (Triticum aestivum L.) pure lines. The effects of particular stages of cryopreservation protocol on water content, number of calli recovering growth, and rate of morphogenesis were analyzed. Regrowth was observed in 90.5–100% of calli after dehydration and in 93.3–100% after freezing-thawing. Dehydration, but not freeze-thawing significantly decreased the frequency of morphogenetic variation.