Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

Background

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.

Results

The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.

Conclusions

These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.     

Development of a gene silencing DNA vector derived from a broad host range geminivirus

Background  

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.     

茄子SmMsrA基因VIGS表达载体的构建及表达分析

利用RT-PCR从茄子单性结实品系D-10中获得甲硫氨酸亚砜还原酶A基因(Sm Msr A)的编码区。通过Gateway同源重组技术,构建了3个分别含Sm Msr A基因不同片段的烟草脆裂病毒(Tobacco rattle virus,TRV)介导的基因沉默(VIGS)表达载体p TRV2-Sm Msr Ai。表达载体转入农杆菌GV3101后,用注射压迫法侵染茄子叶片。采用表型观察、TRV病毒分子检测和q RT-PCR技术,评价构建的VIGS体系对Sm Msr A基因的沉默效果。结果表明,报告基因PDS沉默后叶片呈现明显的光漂白现象,Sm Msr A基因沉默后叶片呈花叶状,果实变小,叶片和果实中的Sm Msr A基因的表达量明显降低,表明Sm Msr A基因是正向调控茄子果实发育的相关基因。     

An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars

Background

Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation.

Results

Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected.

Conclusions

The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.     

Phenol induced browning and establishment of shoot-tip expiants of 'Fuji' apple and 'Jinhua' pear cultured in vitro

SUMMARY

Several factors influencing phenol induced browning and establishment of apple (Malus pumila Mill. cv. Fuji) and pear (Pyrus bretschneideri Rend., cv. Jinhua) were investigated by using shoot-tip expiants cultured in vitro. Expiants of 'Fuji' apple and 'Jinhua' pear collected from November to February produced low browning percentages. Browning percentages increased rapidly as the stock plants entered the growing season, reached a maximum during April and August and then decreased. Dark treatment of the stock plants reduced browning. Lowest incidence of browning and the highest surviving percentages of both 'Fuji' apple and 'Jinhua' pear were obtained after four weeks of dark treatment. However, bud-break percentage, fresh weight and the number of new leaves of the surviving expiants decreased with increase in the length of dark treatment. When the dark treatment was used during the first phase of culture initiation, 5°C was better than 24°C for reducing browning and improving establishment of the expiants. At 5°C, 6 d of dark treatment for 'Fuji' apple and 8 d for 'Jinhua' pear produced the least browning and the highest surviving percentages. However, bud break percentages, fresh weights and numbers of new leaves decreased as the length of the dark treatment increased. Addition of 2.5 g l"1 activated charcoal to the initiation medium favoured establishment of 'Jinhua' pear expiants and 100/150 mg 1"' ascorbic/citric acid were effective with 'Fuji' apple.     

Einfluss rezenter Klimaveränderungen auf die Phänologie bei Kernobst am Standort Klein-Altendorf – anhand 50-jähriger Aufzeichnungen

This project examines the effects of climate change on pome fruit phenology at Klein-Altendorf in the Rhineland fruit-growing region in the West of Germany, using 50 years of weather data and phenology records, including beginning of flowering (F1), full bloom (F2), harvest date and leaf drop of apple and pear, with the following results:

1. Fifty years of weather records and pome phenology data showed a distinct separation between a first phase (1958–1987), which was 0.42°C cooler, and a second phase (1988–2007), which was 0.66°C warmer than the long-term, 50 year temperature average of 9.4°C, indicating the start of the climate change in 1988.
2. Annual average temperatures rose from 8.8°C to 10.2°C, i.e. by 1.4°C over 50 years at Klein-Altendorf, which affected the apple phenology. A comparison of the second phase (average temp. 10.1°C) with the first phase (9.0°C) resulted in a temperature rise of 1.1°C; the temperature rise in the winter (1.2°C) was greater (by 0.2°C) than in the summer (1.0°C). This temperature rise in the last 20 years (second phase, 1988–2007) was more pronounced in the winter months January ( $ + $ 1.7°C) and February ( $ + $ 1.8°C) than in the spring months March and May both with $ + $ 1.4°C leading to enhanced early post-bloom fruit drop, and July ( $ + $ 1.3°C) and August ( $ + $ 1.6°C).
3. Annual precipitation remained constant at ca. 600?mm with an increase in February, March and April and an equivalent decrease in May (?10?mm) and July (?7?mm) during fruit development.
4. Soil temperatures increased by 2.6°C and hence greater than the air temperature ( $ + $ 1.45°C) over the last 50 years; this increase was most pronounced between January and March.
5. Minimum air temperatures increased also by 2.6°C from ?6.1°C in March 1958 to ?3.5°C in March 2007. The more advanced flowering, brought about by warmer January to March temperatures, now coincides with late spring frosts, thereby maintaining the risk of yield loss due to spring frost in April, a month when the minimum temperature still continues to drop.
6. On average, apple and pear flowering was 10 days earlier without extension of the flowering period after warm winters and appeared more severely affected than the harvest period (3–9 days) and leaf drop (2–3 days earlier) with distinct varietal differences. The apple flowering period was 4 days shorter in the last 10 years and reduced from 12–15 days to 8–10 days. Early maturing cultivars (cvs) ripened 3–4 days earlier, while late cvs ripened 8–12 days earlier. The period between harvest and leaf drop was extended by 2–7 (apple) to 11 days (pear), which requires regional differentiation according to the respective climatic zone and may be beneficial for nutrient translocation into the perennial woody parts of the tree.
7. Since the changes in the temperature-based vegetation period did not explain the effects of climate change on pome phenology, a `pomological vegetation interval' between the beginning of flowering and beginning of leaf drop was defined and was extended by climate change in spring by 0–10 days in apple and by 8 days in pear. Based on flowering (F1, F2) advances, the Meckenheim fruit-growing region was more strongly affected by the climate change compared with other European fruit growing regions.

     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

Summary

Compared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.     

梨矮化基因pcDw的SSR标记定位

以矮化梨(Pyrus communis L.)与茌梨(P.bretschneideri Rehd.)的F1杂交分离群体共110个单株(3a生,矮化型和正常型各55株)为试材,对来自西洋梨的矮化型突变基因pcDw进行了SSR分子标记研究。用分离群体分组分析法(Bulked Segregant Analysis,BSA),通过对源自梨、苹果和桃基因组的共40对SSR(Simple Sequence Repeat)引物的筛选,获得了一个与pcDw基因连锁距离为9.3cM的SSR标记KA14210,由此将该基因定位到了梨品种Barlett遗传图谱的第16连锁群上。     

Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers

Although there have been some attempts to obtain intergeneric hybrids between Japanese pear (Pyrus pyrifolia Nakai) and apple (Malus × domestica Borkh.) mature hybrids have not been generated due to hybrid lethality. Shoots from immature hybrid embryos were gamma irradiated and cultured at normal temperature conditions to obtain viable intergeneric plants. Gamma irradiation doses of 50 and 100 Gy cultured at 25 °C killed all shoots but there was one survivor at 150 Gy and 25 °C. Hybrid status of the shoot was confirmed by using morphology, flow cytometry and SSR markers. The intergeneric plant had similar leaf serrations to apple and similar petioles to Japanese pear. Flow cytometric analysis revealed that the intergeneric shoot had a DNA content intermediate between the parents. SSR analysis showed one allele from each parent for all 16 SSR loci investigated. These data strongly suggest that the obtained shoot is a true hybrid between Japanese pear and apple.     

Beschreibung des Feuerbrandbefalls am Institut für Obstzüchtung in Dresden-Pillnitz im Jahr 2003

The orchard of the Institute of Fruit Breeding of the German Federal Centre of Breeding Research on Cultivated Plants in Dresden-Pillnitz was highly affected by fire blight in 2003. Infected pomefruit trees were observed over a period of nearly 3 months. The first symptoms on pear trees were found on May 19th. The pathogen Erwinia amylovora was confirmed officially on May 26, and the last infected apple trees were detected the 11th of August. The infected trees had to be grubbed at the decision of the Phytopathological Authority. In total, 1164 apple and 478 pear trees were grubbed, including the entire pear collection of the gene bank. Of 35 wild species of pear, 49 accessions, eight accessions of six species each, showed infections. The apple collection of the gene bank included 33 wild species, with 365 accessions, and 845 cultivars and clones. Ten accessions of nine wild apple species and 81 cultivars/clones of these collections showed fire blight infection. The source of infection was the pear collection, and the distance from that source was important for the occurrence of infection. Field plots close to the pear collection had tree losses of 10–34%, while more distant plots had losses of 0–6%. Around 80% of the lost apple trees were detected and grubbed from 27th May to 11th June. Some of the cultivars bred in Dresden-Pillnitz, e.g. ‘Pilot’ and ‘Rekarda’, were affected by fire blight in most field plots, whereas most others were affected mainly only in plots adjacent to the infection source. A correlation of r=?0.72 could be calculated for rating in artificial shoot inoculations and percentage of trees of resistant cultivars lost. The cultivars ‘Pirol’, ‘Reanda’, ‘Remo’, ‘Rene’, ‘Renora’, ‘Resi’, and ‘Retina’ showed only a very low numbers of infected trees. No tree of ‘Rewena’ showed symptoms of fire blight. Despite a tendency to postblooming, only 8.9% of ‘Pinova’ trees had to be grubbed.     

Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants

Background  

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.     

Evaluation of alternative RNA labeling protocols for transcript profiling with Arabidopsis AGRONOMICS1 tiling arrays

Background

The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.

Results

We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.

Conclusions

The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.     

Obstkonsum in China �C Anbau, Angebot und Preise

As a Northern hemisphere fruit producer, China??s harvest time overlaps with that in Europe and the US and has been dominating the worldwide fruit market. Prices and sources of fruit in China were examined in supermarkets in Beijing in May 2011 and combined with the official Chinese Agricultural Yearbook to analyse China??s fruit industry:

1. China has become the world??s no 1 for apple (25?C30 mil. t), pear (13.5 mil. t) and Citrus (23 mil. t) production based on the Chinese statistical agricultural yearbook (2009), and no. 3 for strawberry (0.65?C0.8 mil. t), with an overall fruit production of 105?C113 mil. t/year and a tendency to increase domestic fruit production and consumption.
2. Fruit acreages, reported in 2009, were 2 mil. ha for apple, 1 mil. ha for pear (inclu-ding Nashi) and 2 mil. ha for Citrus and 0.083 mil. ha for strawberry totalling 10.7 mil. ha for fruit production in China, with a tendency towards slightly decreasing orchard acreage. The average farm size was ?£?0.5 ha.
3. Fruit consumption in China was calculated as 18?C21 kg apple/head/year, 10.1 kg pear/head/year (incl. Nashi) and 77?C81 kg fruit/head/year based on 1.33 billion inhabitants and production data; this is a rise in domestic fruit consumption from 45 kg in 2002/2003 to 77?C83 kg fruit/head/year in 2008/2009.
4. Jujube fruit yielded a farm gate price of 1.20 ?/kg and cv. ??Fuji?? apple 0.55 ?/kg (5 Yuan) as for most pome and stone fruit (with higher prices in the farm shop), which doubled to 1.10 ?/kg after sea export from Qingdao harbour to Rotterdam.
5. Prices for Chinese cv. ??Fuji?? apples in Beijing supermarkets ranged from ? 0.84/kg for a poor class II to ? 2.80/kg for class I fruit. Three (??Red Delicious??, ??Gala?? and ??Granny Smith??) out of five apple varieties on sale out-of-season in China??s Capital were imported from the US or Chile, with higher prices ranging from ? 3.25 for imported cv. ??Gala??, 4.15 ?/kg for ??Red Delicious?? to ? 4.90 for cv. ??Granny Smith??.
6. The Chinese viz Asian taste prefers large, juicy and sweet, red or yellow fruit with little acidity and smooth skin viz peel finish. Surprisingly, ??Granny Smith?? apples were sold as ??green apples?? without variety name at the highest price of all apple varieties.