Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in ‘Elstar’ apple and the effect of grafting

Summary

Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple.Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus × domestica Borkh., ‘Elstar’) with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type ‘Elstar’, two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages.     

Impact of controlled atmosphere storage conditions on storability and consumer acceptability of sweet cherries ‘Regina’

Summary

Sweet cherries obtained from a rain-protected orchard were subjected to various controlled atmosphere storage conditions for up to seven weeks. Quality parameters responded differentially to various CA-conditions: changes in fruit firmness, sugar content and colour were mostly not significant. However, the storage conditions exerted significant impact on other parameters such as respiration rate, RQ, ATP- and ADP-concentrations. Air-stored fruits respired at significant higher rates than CA or ultra low oxygen-stored fruits. Moreover, the impact of CA-storage conditions persisted even after a conditioning period of 36 h at room temperature. The ATP and ADP concentrations of sweet cherries behaved similarly, where a combination of reduced O₂ and very high CO₂ concentration (12% or above) resulted in highly reduced ATP-concentration in the fruit tissue. A taste panel considered fruits stored under 6% CO₂ + 2% O₂ as good, although there was a strong indication that it is better to store sweet cherries for a limited time (up to five weeks) under a low O₂ concentration combined with a high CO₂ concentration (12%) or under a high O² concentration combined with a very high CO₂ (18%).     

Effects of rate of establishment of storage conditions and ethylene removal on the storage performance of ‘Cox's Orange Pippin’ apples

The use of rapid establishment of temperature and CA storage conditions, as well as ethylene removal to reduce loss of firmness and ground colour, during storage of apples (Malus domestica Borkh, ‘Cox's Orange Pippin’) in <1% CO₂ + 1.25% O₂ at 3.5°C was investigated. Samples subjected to rapid establishment (RE) of storage conditions had a higher peel chlorophyll content after 5 months storage, but not after 8 months, than samples from containers where the conditions had been established at a rate typical of normal, well-maintained commercial stores (CR). RE samples were considered firmer than CR by a sensory panel in February but not in May, while penetrometer readings were the same on both occasions. RE samples which had additionally been subjected to a pre-harvest daminozide spray and ethylene removal during storage (LE) were firmer, had a higher peel chlorophyll content and, on the green parts of the surface, a lower Hunter “b” value in February than both CR and RE samples. The effects of LE on chlorophyll and ground colour were maintained until May, but in both February and May only the ground colour effect was maintained during a simulated marketing period of 14 days at 10°C in air following storage.     

苹果Mal d 1基因的原核表达及纯化

以"王林"苹果为试材,采用RT-PCR的方法克隆获得Mal d 1基因,并将该基因连接到原核表达载体pColdⅡ上,经测序确定所构建的重组载体pCold-Mal d 1开放阅读框正确。将获得的重组质粒pCold-Mal d 1转化大肠杆菌BL21(DE3)菌株,经IPTG 0.1mmol·L~(-1),15℃,24h冷诱导表达,SDS-PAGE电泳检测,证明Mal d 1蛋白获得了稳定而高效的表达,所表达蛋白是大小约为22.4kD的融合蛋白。经镍柱纯化后获得相对单一的蛋白,可用于免疫组织化学,蛋白印记检测等。     

Prohexadione-calcium effects on the quality of ‘Royal Gala’ apple fruits

Summary

To study the effects of prohexadione-calcium (Pro-Ca) on fruit quality, an experiment was performed in 2002 in which trees were treated with two different concentrations of Pro-Ca and left with different crop-loads. Increasing levels of thinning resulted in smaller yields, but greater fruit sizes, which followed a curvilinear pattern when plotted against crop-load. The soluble solids content and titratable acidity of fruits decreased linearly with increasing crop-load. Fruit skin chromaticity values also changed. Luminescence increased, while a* values decreased linearly with increasing crop-load, indicating more reddish and darker coloured fruits on trees subjected to very heavy thinning. In addition, the concentrations of soluble sugars in fruits changed with the level of cropping; fructose and glucose concentrations increased with crop-load, while sorbitol and sucrose decreased. The application of Pro-Ca shortly after full-bloom inhibited shoot elongation growth, but did not result in changes in yield or in any of the fruit quality parameters analysed.     

Improvement of fruit quality and storage potential of ‘Braeburn’ apple through deficit irrigation

Summary

We explored the potential of deficit irrigation (DI) applied at different times during the growing season for improving fruit quality and storage potential of ‘Braeburn’ apple growing in a commercial orchard. Because DI often reduces fruit size, effects of fruit size on fruit quality were also examined. The irrigation treatments were: commercially irrigated control (C), early deficit irrigation (EDI) applied from 63 to 118 d after full bloom (DAFB), and late decifit irrigation (LDI) applied from 118 DAFB to final harvest on 201 DAFB. Both EDI and LDI improved fruit quality in terms of increases in: dry-matter concentration (DMC) at harvest, flesh firmness, total soluble solids, and total soluble sugars both at harvest and after storage. The DI fruit had less weight loss during storage than did C fruit. Deficit irrigation affected the concentrations of a few individual aroma volatiles but not total volatile concentration. Incidence of physiological disorders was not affected by irrigation treatments. Mean fruit weight was lower in DI than in C but the difference was not significant. Among the quality attributes studied, only firmness and DMC were affected by fruit size with their values being higher in smaller fruit.     

The effect of summer pruning and nitrogen on growth,cropping and storage quality of Cox’s Orange Pippin apple

Pruning in late summer was compared with winter pruning on mature 23-year-old spur pruned trees of Cox’s Orange Pippin on M.2 rootstock. Two nitrogen treatments were superimposed, namely a standard dressing of 126 units N (sulphate of ammonia) applied in March as compared with 288 units N (nitrate of soda) applied in mid-August at the time of summer pruning. Summer pruning reduced trunk girth increment, advanced blossom development, increased the amount of red colour on the fruits and drastically reduced the incidence of bitter pit in stored fruit. The treatments did not affect cropping. Nitrogen treatments had no overall effect upon the incidence of bitter pit, but in the first season, when bitter pit was most prevalent, high levels of N applied in August reduced the amount, a similar trend being recorded the following year.     

Effects of different rootstocks and training systems on the mineral composition of ‘Delicious’ apple leaves

Leaf mineral composition as affected by different rootstocks, cultivars, training systems and their combinations was measured during 1987 and 1988. Trees on M.7 had significantly higher concentrations of N, P and Fe, whereas those on MM. 106 maintained higher values of K, Ca, Mg and Mn during both years. Between cultivars, Red ‘Delicious’ showed greater accumulation of N, P, Mg and Fe but K and Cu levels were found to be more in Starking ‘Delicious’. All mineral nutrients except Ca and Mg accumulated more in spindle-bush trained trees than those under modified central leader system. The Mineral composition of Starking ‘Delicious’ grafted on M.7 rootstock and trained as a spindle bush produced the best yield efficiency and fruit quality, and fell within the normal range.     

抗苹果斑点落叶病基因Mal d 1的克隆及功能鉴定

以苹果叶片总RNA为模板,通过克隆获得抗苹果斑点落叶病基因Mal d 1。Mal d 1开放阅读框长度为480 bp,编码159个氨基酸残基,包含2个外显子和1个内含子。蛋白结构分析显示,Mal d 1蛋白包含bet v 1-like结构域。荧光定量PCR分析结果表明,Mal d 1在苹果叶、花、果皮、果肉中均有表达,但各器官中表达水平存在差异;在不同发育时期的果皮中都有表达,表达水平随时间呈现先上升后下降的趋势;在抗性品种叶片中表达水平较高;在叶片人工接种病菌条件下表达量随时间的延长明显高于对照组。利用PRI101-AN载体和农杆菌介导的遗传转化方法转化苹果愈伤组织,转基因愈伤组织的抗病鉴定结果显示,Mal d 1过量表达增强愈伤组织对苹果斑点落叶病的抗性。     

Effects of controlled atmosphere,edible coating,and 1-methylcyclopropene on improving storage quality of ‘Bartlett’ pears after long-term storage

Recent trends towards greater fresh market use of ‘Bartlett’ pears has increased the need to extend its storage life to prolong the packing and marketing season in the United States Pacific Northwest region. Sixteen and 38%, respectively, of control fruit developed senescence disorders following 5 and 6 months of storage at ?1.1°C. Commercial standard controlled atmosphere (CA) conditions (O₂ at 1.5 kPa and CO₂ < 1 kPa) or edible coating (Semperfresh?, SF) prevent the appearance of senescence disorders for 5 months, but 9% and 16% of fruit, respectively, developed senescence disorders after 6 months. The combination of CA+SF completely inhibited senescence disorders for 6 months. Treatment with CA and SF, alone or in combination, maintained high-storage quality and developed ripening capacity with characteristic melting texture during storage. Senescence disorders were inhibited for 6 months by 0.3 µL l^?1 1-methylcyclopropene (1-MCP), alone or combination with CA or CA+SF. In part these pears developed ripening capacity after 6 months of storage. The combination of CA+SF+1-MCP maintained the highest storage quality with dark green colour and hard firmness, which might be associated and proportional with reductions in ethylene synthesis and respiration rate after long-term storage.     

Evaluation of the activity of aqueous extracts of some organic waste materials on in vitro-cultured shoots of ‘M9’ apple rootstock

Summary

The present work investigated the effects of different aqueous extracts of organic waste compounds on growth, proliferation and photosynthetic activity in ‘M9’ (Malus domestica Borkh.) shoot cultures, with the aim of determining the feasibility of using in vitro cultures as a tool for the rapid evaluation of organic amendments in agriculture. Aqueous extracts of the following organic waste compounds: cow manure (CM), sugarbeet industrial waste (SB), mixed grape, poultry and municipal solid waste (GPM), and citrus pruning and industrial waste (CPI) were prepared at a rate of 1:10 (w/v) compound:distilled water. The basal media used in the proliferation phase were: (i) PM1, modified Murashige and Skoog (MS) enriched with 4.4 µM 6-benzyladenine (BA); (ii) PM2, as PM1 but with a reduced cytokinin concentration (1 µM BA) to evaluate possible hormone effects; and (iii) PM3, 4.4 µM BA with reduced salt strength (0.33 MS) to induce nutrient deficiency. Hormone-free medium with half-strength MS salts was used for rooting. All media were enriched with each extract at 0, 0.2, 2, 20 or 200 ml l^–1. Photosynthetic activity was measured on PM3 medium enriched with SB or CM. Standard culture conditions were 22° ± 2°C, with a 16 h photoperiod at 30 µmoles photosynthetically active radiation (PAR) m^–2 s^–1, but at 80 µmoles PAR m^–2 s^–1 to determine photosynthetic activity. Shoot weight increase in PM1 was not affected by the GPM and CPI extracts, while the growth trends of CM- and SB-treated shoots were described by a second-degree function with maxima at 2 ml l^–1 and 0.2 ml l^–1, respectively. Shoot proliferation for SB was represented by a quadratic curve (maximum at 2 ml l^–1), was linearly reduced as GPM increased, but was not affected by CM or CPI. Treatments did not significantly affect rooting percentage and root length; however root number was increased by SB at 2 ml l^–1.CO₂ fixation increased linearly with both SB and CM, despite reduced growth at the highest levels of extract.     

The thinning effect of benzyladenine on red ‘Fuji’ apple trees

A trial, at the Grove Research Station in southern Tasmania, studied the thinning effect of benzyladenine (BA) on five year old red ‘Fuji’ apple trees. Untreated control trees were compared with trees treated at four timings, from full bloom (FB) to 30 d after full bloom (AFB), with four concentrations of BA (50, 100, 200 or 400 mg I-1). The most effective time of thinning was 20 d AFB, when increasing concentration also increased thinning. Further advantages of this successful thinning were reflected in better fruit size and weight. At 30 d AFB high concentrations of BA depressed fruit weight. Return bloom was improved significantly by both 20 and 30 d. AFB sprays with a concentration effect at 20 d AFB. Timing of sprays had no effect on the incidence of russet which was increased only by applications of 400 mg l-1. Both the higher concentrations and later timings resulted in an increase in shoot numbers and total growth. Reduction in mean shoot length was related more to the concentration of BA used than to differences in the timing of sprays. BA has shown potential as a thinner for red ‘Fuji’ apple at 20 d AFB.     

The effect of gibberellins and cytokinins on fruit development of Bramley’s Seedling apple

Two applications of a 1 : 1 mixture of gibberellins A₄ and A₇ to either frost damaged or artificially decapitated Bramley's Seedling apple flowers resulted in increased fruit set and yield. It also increased the crop in an orchard without pollinators. Gibberellic acid was not effective. The addition of cytokinins had no beneficial effects and caused malformation.     

The interaction of Cytolin and NAA on cropping red ‘Delicious’ apple

A trial, at Spreyton in north-western Tasmania, studied the effects of Cytolin and NAA sprays on the thinning and shape of ‘Delicious’ apples. Untreated control and hand thinned trees were compared with trees treated with Cytolin or NAA alone, or in combination. Cytolin was applied at 25 mg 1~‘ at the king petal (KP) stage. NAA was applied at 8 mg I“1 at full bloom (FB) and 7,10, or 15 days after full bloom (AFB). Cytolin alone had no effect on thinning, fruit weight or on pip numbers but it did improve fruit shape. The combination of cytolin and NAA at FB overthinned. Cytolin and NAA combinations applied at all times after FB significantly decreased both mean fruit weight and pip numbers and reduced the percentage of fruit > 70 mm. All Cytolin-NAA combinations and the NAA treatments applied at 10 and 15 days AFB resulted in unacceptably high percentages of apples < 45 mm in diameter (pygmy fruit). It is recommended that Cytolin and NAA should not be used in the same spray programme. NAA alone is a most effective thinner at FB or seven days AFB.     

Does manipulation of fruit-tree architecture affect the development of pests and pathogens? A case study in an organic apple orchard

Summary

Centrifugal training, based on the extinction procedure, is an innovative type of apple tree training recently used in French apple orchards. A thinning cut of fruiting spurs along the trunk and on the proximal and underside parts of the fruiting branches modifies the within-tree leaf density and light environment, which is likely to affect the development of some apple pests and pathogens. In this study, development of the most serious foliar pests (aphids, mites) and disease (apple scab) was assessed over 3 years in an organic apple orchard with two training systems: the centrifugal training (CT) system, and the Original Solaxe (OS) system, using a bent axis with no removal of fruiting spurs, as a control. Infestation levels were significantly lower in 2002 and 2004 in the CT system than in the OS system for the most prevalent pest, the rosy apple aphid, Dysaphis plantaginea and, in 2003, for the red spider mite, Panonychus ulmi. Infestation by the green apple aphid, Aphis pomi, was higher in June 2002 and 2004 and lower in June 2003 in the CT system, compared to the OS control. The incidence of apple leaf scab was lower in the CT system than in the OS control in Spring 2002 and Spring 2004; but, later in the Summer, it increased more in CT trees, resulting in no difference between training systems in the levels of fruit infection at harvest. Hypotheses considered were based mainly on: (i) the removal of pests or inoculum due to the thinning cut of fruiting spurs for CT trees; (ii) effects on within-tree microclimate, providing a more aerated canopy in the CT system; (iii) shoot density and distribution, allowing greater distances between growing shoots in CT trees; and/or (iv) the shoot growth dynamics, allowing the presence of attractive or susceptible leaves in late June. Since the most serious apple pests and diseases were less prevalent in the CT system over the 3 years of the experiment, CT may prove to be a complementary, sustainable means to contribute to crop protection, especially in organic or integrated pest management orchards.     

What determines the spatial extent of landscape effects on species?

Context

Landscape ecologists are often interested in measuring the effects of an environmental variable on a biological response; however, the strength and direction of effect depend on the size of the area within which the environmental variable is measured. Thus a central objective is to identify the optimal spatial extent within which to measure the environmental variable, i.e. the “scale of effect”.

Objectives

Our objectives are (1) to provide a comprehensive summary of the hypotheses concerning what determines the scale of effect, (2) to provide predictions that can be tested in empirical studies, and (3) to show, with a review of the literature, that most of these predictions have so far been inadequately tested.

Methods

We propose 14 predictions derived from five hypotheses explaining what determines the scale of effect, and review the literature (if any) supporting each prediction. These predictions involve five types of factors: (A) species traits, (B) landscape variables, (C) biological responses (e.g. abundance vs. occurrence), (D) indirect influences, and (E) regional context of the study. We identify methodological issues that hinder estimation of the scale of effect.

Results

Of the 14 predictions, only nine have been tested empirically and only five have received some empirical support. Most support is from simulation studies. Empirical evidence usually does not support predictions.

Conclusions

The study of the spatial scale at which landscape variables influence biological outcomes is in its infancy. We provide directions for future research by clarifying predictions concerning the determinants of the scale of effect.

     

Efficacy of Natureseal® AS1 browning inhibitor in fresh-cut fruit salads applications,with emphasis on apple wedges

Summary

Prevention of browning in fresh-cut fruit is of major commercial significance as fresh-cut fruit salads are major players in the international marketplace. Natureseal^® AS1 and AS5 browning inhibitors out-performed ascorbic acid and citric acid in tests with fresh-cut wedges of ‘Bramley’ apple. A dipping regime of 2 min in 6% (w/v) AS1 solution was found to be optimal, as longer dip-times and higher concentrations led to significant residues of AS1 on the fresh-cut ‘Bramley’ wedges. Tests on fresh-cut wedges from ten apple cultivars indicated that AS1 was significantly beneficial (P < 0.001) to maintain wedge colour during chill-storage in all cultivars, with the exception of ‘Shampion’, which had a low browning tendency and performed equally well with or without AS1. Tests on the anti-oxidant status of a generic fruit salad, which was not treated with AS1 and stored for 15 d, indicated a significant reduction in anti-oxidant status, due largely to the depletion of vitamin C (from 137 to 97 mg 100 g^?1 DW). Total phenolics contents remained relatively constant over the 15-d storage period. The outcomes from these trials have been presented to commercial food companies with a view to their uptake.     

Evaluating the effectiveness of different strategies for calcium application on the accumulation of calcium in apple (Malus × domestica Borkh. ‘Braeburn’) fruit

Summary

Orchards displaying calcium (Ca) deficiency are a common phenomenon worldwide, despite the presence of sufficient Ca in the soil and the plant. A 3-year trial was conducted between the 2007 – 2008 and 2009 – 2010 growing seasons to evaluate the contributions of soil and foliar Ca applications to Ca concentrations in ‘Braeburn’ apple (Malus × domestica Borkh.) fruit. Ca(NO₃)₂ (Calflo; Yara Africa, Fourways North, South Africa) was applied as six separate foliar sprays until run-off. Applications were made at 1-week intervals between approx. 21 – 70 d after full bloom (DAFB) at 6.75 ml l^–1. Soil applications of Ca (Tropicote^TM; International ASA, Oslo, Norway) at 300 kg ha^–1 were applied at fruit set, or after harvest, according to standard practice. Mineral analysis was conducted to assess the soluble Ca concentrations of whole fruit (without pips and stalks), to quantify the contribution of foliar sprays or soil-applied Ca. Fruit Ca concentrations were maintained at satisfactory levels (4.5 mg Ca 100 g^–1 FW) at harvest by applying a series of six foliar sprays early in the season (for all seasons) during the trial period. Fruit Ca concentrations at 80 DAFB were highest in the treatments with foliar applications of Ca. In 2009 – 2010, Ca concentrations in apple fruit were lowest (8.38 mg 100 g^–1 FW) for soil application of Ca at fruit set. Ca applications to soil after harvest in the previous season, and soil applications shortly after fruit set in the current season, did not significantly increase Ca concentrations in current-season fruit, providing soil Ca levels were above the minimum requirement for apple trees. A possible explanation is that apple trees regulate their uptake of Ca through the roots when soil Ca is available in sufficient quantities. This confirms the importance of active root growth for efficient Ca uptake by apple trees when applying Ca to the soil.     

Influence of temperature on seed germination in papaya under subtropical conditions of India

The effect of temperature on germination and days taken for germination was evaluated in Carica papaya cvs., ‘Pune selection-3’ and ‘Pusa Dwarf’. The highest average seed germination per cent was observed in the month of July (96.8 and 93.0?%), followed by August (94 and 88.5?%), whereas the lowest average seed germination was observed in the month of January (44.2 and 36.8?%) in ‘Pune selection-3’ and ‘Pusa Dwarf’, respectively. Minimum number of days (5.17 and 6.33) was observed for germination in the month of July, whereas the maximum number of days (29.5 and 31) was taken in the month of January in both the cultivars, respectively. The average highest germination and seedling height was observed at temperature 29.8 °C and 29.5 °C during July and August, respectively, whereas the lowest was observed at temperature 14 °C and 15.9 °C during January and December, respectively. The optimum time for nursery sowing was July to August for autumn season crop under subtropical conditions of India.