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 storage conditions on the apple allergen Mal?d?1

Consumption of fresh apple fruits can induce allergic reactions in humans. The apple allergen Mal?d?1 is responsible for these allergic reactions in humans in Central Europe and North America. Biosynthesis of Mal?d?1 depends on apple cultivar, and its concentration increases with time during fruit storage. However, data on the impact of different fruit storage conditions during long-term storage are scarce. Hence, the Mal?d?1 contents of eight apple cultivars were analyzed for this study during long-term storage in a cold chamber as well as under controlled atmosphere conditions (CA). After harvest, apple fruits were stored for 12, 20, 28 or 36 weeks in a cold-chamber at +?2?°C or as under controlled atmosphere conditions of 1.5?% CO₂, 1.5?% O₂ at +?2?°C. Mal?d?1 content in apple fruit of all eight cultivars examined increased during fruit storage. In most cases, differences between Mal?d?1 of apple fruits stored in the cold chamber and under CA conditions were significant, but inconsistent. In apple cv. ??Elise??, fruits stored in the cold chamber contained more Mal?d?1 compared with those stored in CA, whereas the situation reversed in other varieties like cv. ??Boskoop??. The greatest Mal?d?1 content was measured in fruits of cvs. ??Golden Delicious?? and ??Gala??, whereas the smallest Mal?d?1 level was in cvs. ??Elise?? and ??Pinova?? over the whole storage time. Overall, this experiment showed the complexity of the relationship between the Mal?d?1 content, allergenicity of apple fruits, different cultivars, storage conditions and storage time. Persons allergic to apple fruits should consume the fruits as fresh as possible or only after a limited storage time. Furthermore, they should prefer apple varieties with a low content of allergenic proteins, such as cv. ??Elise?? or cv. ??Boskoop?? as a cultivar known for its large polyphenol content.     

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus × domestica Borkh.)

Summary

The conventional selection system for apple transformation is based on the selectable marker gene, nptII, encoding antibiotic resistance against kanamycin. We tested an alternative selection system based on the use of D-amino acids using the gene, D-amino acid oxidase 1 (dao1) as the selectable marker, in order to avoid the presence of antibiotic resistance genes in the resulting transgenic apple plants. In addition, dao1 allowed the selection as well as the elimination of dao1-transgenic plants, based on differences in the toxicity of different D-amino acids. Regeneration experiments using apple leaf explants revealed that 2 mM D-serine or D-alanine inhibited shoot regeneration. We performed transformation experiments using the apple cultivars ‘Gala’, ‘Holsteiner Cox’, and a progeny of the apple cultivar ‘Pinova’, and the vector p35S::dao1-intron, containing the dao1 and nptII selectable marker genes. Several shoots regenerated successfully on selection media containing various concentrations of D-serine or D-alanine, but transgenic shoots were not obtained. However, three dao1/nptII transgenic apple lines were obtained after selection with kanamycin, indicating that the vector was functional. Furthermore, we showed that 20 mM D-serine could be used to select dao1-transgenic shoots from non-transgenic in vitro shoots, whereas 13 mM D-isoleucine had the opposite effect.     

Effects of 1-aminocyclopropane carboxylic acid on the rate of ethylene release from detached fruiting spurs and on fruit abscission in apple

Summary

1-Aminocyclopropane carboxylic acid (ACC) was applied to apple (Malus × domestica Borkh.) trees or to individual fruiting spurs between full bloom (FB) and 31 d after full bloom (DAFB; mean fruit diameter of 20 mm) to determine the effects of ACC on the ethylene released from detached spurs and on fruit abscission. The rate of ethylene release from detached fruiting spurs increased 1 d after applying 50 mg l^–1 ACC at full bloom or 16 DAFB (mean fruit diameter of 10 mm), but ACC had a reduced effect on the rate of ethylene release when it was applied 31 DAFB (mean fruit diameter of 20 mm). ACC reduced fruit set in ‘GoldRush’ apple in a concentration-dependent manner in 2009 and in 2010, but had no thinning activity on ‘Pink Lady’ in 2010. ACC at 200 mg l^–1 resulted in the yellowing and abscission of ‘GoldRush’ spur leaves in 2009, but not in 2010. The effects of combining ACC and 5 mg l^–1 naphthaleneacetic acid on fruit set in ‘GoldRush’ apple were additive. The application of 200 mg l^–1 ACC when fruit are ca. 10 mm in diameter may therefore be a useful chemical thinning treatment for apple fruit.     

Studies on the Maturity of Apples.: Respiration Progress Curves for Cox’s Orange Pippin Apples for a Number of Consecutive Seasons

Summary

The effect of UV-B irradiation at 10°C and 20°C on the quercetin glycosides procyanidins, chlorogenic acid and anthocyanin levels in the skin of detached fruit of five apple cultivars was investigated. UV-B responsiveness was compared between sides of the fruit that were outwardly facing (exposed) and inwardly facing (shaded) when on the tree. There were no common effects of UV-B irradiation and temperature across all cultivars. UV-B irradiation increased the quercetin glycoside levels in the shaded side of ‘Gala’, ‘Royal Gala’ and ‘Braeburn’ fruit and only at 20°C. UV-B irradiation of the exposed sides of the five cultivars, of either side at 10°C, and of ‘Pacific Rose’ and ‘Aurora’ at either 10°C or 20°C had no significant effect on quercetin glycoside levels. There was no effect of UV-B irradiation on procyanidin levels at any combination of treatments. However, levels of chlorogenic acid, although one tenth of those of quercetin glycosides and procyanidins, increased markedly with UV-B irradiation. Both exposed and shaded sides of fruit of all cultivars increased in chlorogenic acid levels at 10°C and 20°C after UV-B irradiation. UV-B irradiation increased anthocyanin levels in exposed and shaded sides of fruit of all cultivars at 20°C, and on ‘Braeburn’, and ‘Aurora’ at both 20°C and 10°C. UV-B irradiation at either temperature had no significant effect on either the relative proportions of the glycosides of quercetin (galactoside, rhamnoglucoside, glucoside, xyloside, arabinoside and rhamnoside) or the relative proportions of the procyanidins (Procyanidins B2, B5, epicatechin and catechin). The effect of UV-B irradiation was variable, depending on cultivar, previous light exposure, temperature and class of flavonoids examined.     

Chlorophyll fluorescence as affected by some superficial defects in stored apples

Summary

Chlorophyll fluorescence, skin coloration and surface contour were measured for apple (Malus domestica Borkh.) fruit with and without common storage and handling surface defects. Defects included C0₂ injury in ‘Empire’, bitter pit in ‘Jonagold’, superficial scald in ‘Cortland’, ‘Law Rome’, ‘Gala’ and ‘Red Delicious’ and bruising in ‘Cortland’. Measurements were made along a transect established on the fruit surface. The fluorescence parameters, minimal fluorescence (Fo), maximal fluorescence (Fm) and photochemical efficiency (Fv/Fm, where Fv = Fm - Fo) were relatively similar around the circumference of undamaged control fruit. Skin colour was not found to influence chlorophyll fluorescence. With the exception of bruises, there were marked changes in fluorescence in the areas where surface disorders or damage had developed. C02 injury and bitter pit reduced Fv/Fm, whereas superficial scald did not. However, scald symptoms were accompanied by a reduction in Fo and Fm. The reduction in Fo and Fm became more marked as scald symptoms intensified during post-storage holding at 22°C. Chlorophyll fluorescence may have some potential for sorting fruit having superficial defects.     

Annual dry-matter production by three apple cultivars at four within-row spacings in New Zealand

Summary

Annual dry-matter production was estimated from eight year old apple trees of ‘Fuji’, ‘Braeburn’ and ‘Royal Gala’ on rootstock M.9, trained as slender spindles, in a spacing trial with trees spaced at 1.3, 1.5, 1.9 and 2.4 m within the row with tree rows 3.5 m apart. Light interception was recorded at intervals during the season. Both fresh weight yield and total dry-matter production were linear functions of light interception, with a higher slope recorded for the two late season cultivars compared with the early maturing ‘Royal Gala’. Comparison of the relationship between mid-season light interception and apple yields with published values from elsewhere, reveals that the slope of the relationship is enhanced under New Zealand conditions. The relationship suggests an upper limit of yield of 169 t ha^–1 at 90% light interception, slightly higher than the maximum yield recorded of 163 t ha^–1. Energy conversion of intercepted light over the growing season into dry-matter production was similar to that observed in the United Kingdom, suggesting that the major climatic advantage in New Zealand over the United Kingdom is the higher growing season radiation integral. Dry matter was allocated in the order fruit>woody tissue>leaves, with partitioning to the fruit of 74, 72 and 63% for ‘Braeburn’, ‘Fuji’ and ‘Royal Gala’, respectively. Under New Zealand conditions, sustainable 70% harvest index values may be achievable for mature apple trees on dwarfing rootstock with late maturing cultivars.     

Apfel oder Acai: Brasiliens Obstbau im ökonomischen und klimatischen Wandel

In times of recession with a 7–10% inflation rate, drop in consumption and loss in value of the Real currency, Brazil as the third largest fruit producer with 46 mil t fruit on 2.2 mil ha (2013), exports ca. 3% of its production worth ca. 765 mil (2013) – 807 (2014) US $, of which 80% is destined for Europe – the fruit export target for the future is 1 bil US $. Apple (cv. ‘Gala’ and ‘Fuji’) production under Southern hemisphere conditions (26–31°S) in Brazil rose from 0.6 mil t in 2002, peaked at 1.4 million t on 39,600?ha in 2011/12, declining in 2012/13 to 1.05 mil t due to orchard reductions in Fraiburgo as a result of lack of chilling, 1.16 mil t in both 20131/4 and 2014/15 and then 0.8 mil t in 2015/16 on 36,090?ha, resembling average apple yields in Germany on 31,400?ha.Apple orchards are essentially free of fire blight and codling moth, but with 1700?mm annual precipitation, they are affected by the scab and Glomorella fungi. Climate change affects apple production: Lack of chilling, caused by cold winters, induces vertical vegetative branches, flat speckled fruit with long pedicels (fruit stalks; cv. ‘Gala’), sunburn (cv. ‘Fuji’). Warm autumns result in a lack of fruit colouration in both varieties and prevent the cultivation of high chill apples, pears and plums.Three major fruit, orange (7.5?kg), banana (6.8?kg) and apple (cvs ‘Gala’ and ‘Fuji’; 4.2?kg apples/head/year; 2014) dominate the overall fruit consumption of 31.4?kg fruit/head/year compared with 36?kg beef, 34?kg pork and ca. 10?kg poultry resulting in ca. 80?kg meat/head/year in Brazil.In the last three years, a new range of storable apple varieties like ‘Venice’ and ’Daiane’ as well as ‘M 58/07’ and ‘M 10/09 – these latter two, still without a variety name – have been, like ’Eva’ added to the existing new breeds from EMBRAPA as very early variety (harvest in January; low chill; 150 CH) and to the summer varieties ‘Condessa’, ’Monalisa’ and ’Princesa’ (all 300–450 CH) without storability; red mutants of ‘Gala‘ (‘Gala, Maxi’ from RASIP) and of cv. ‘Fuji’ (‘Fuji Suprema’) both with 500–600 CH have been bred for growing in Brazil’s higher altitudes. The new cultivars provide medium-sized fruit, bright red peel colour and sweet taste with little acid and a sugar: acid ratio of 25–55:1, as required by Brazil’s domestic market, but so far lack market acceptance and a marketing concept.     

Chemical composition of French and Polish cloudy apple juices

Summary

Cloudy juices from six apple cultivars from Poland (‘Ariwa’, ‘Gold Milenium’, ‘Florina’, ‘Melfree’, ‘Novamac’, and ‘Rajka’) and four French cultivars (‘Ariane’, ‘Chanteline’, ‘Judeline’, and ‘Judor’) were produced and chemically characterised. The analyses encompassed 23 chemical parameters and phenolics profiles. The most important parameter, differentiating cloudy juice from clear juice, was turbidity. Cloudy juices were characterised by having an average total turbidity of 1,210 Nephelometric Turbidity Units (NTU) and a stability of turbidity of 42%. Some of the results deviated from the accepted ranges given in reference values for apple juices in the Code of Practice of the European Fruit Juice Association. This occurred in the cases of simple sugars and saccharose contents of the Polish apple juices (e.g., up to 56.9 mg l^–1 of saccharose vs. a maximum value of 30 mg l^?1), and for some mineral compounds in the French apple juices (e.g., sodium values up to 10 mg l^?1). Large variations were found in case of important healthconferring components. Water-soluble pectin contents varied from 205 mg l^?1 for ‘Chanteline’, to 1,289 mg l^?1 for ‘Gold Milenium’; while, in the case of phenolic compounds, the range was from 85.7 mg l^?1 for ‘Novamac’, to 524.8 mg l^?1 for ‘Melfree’.     

Application of five DNA marker techniques to distinguish between five apple (Malus × domestica Borkh.) cultivars and their sports

Summary

Genetic variation between five apple cultivars (‘Golden Delicious’, ‘Gala’, ‘Jonagold’, ‘?ampion’, and ‘Idared’) and ten of their sports (‘Golden Delicious Reinders’, ‘Goldrosio’, ‘Gala Must’, ‘Gala Schniga Schnitzer’, ‘Jonagored’, ‘Jonagold Excel’, ‘Szampion Arno’, ‘Szampion Reno Malinowy’, ‘Idaredest’, and ‘Red Idared’) was investigated using five types of DNA markers: Inter-Simple Sequence Repeats (ISSR), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), and Inter-Primer Binding Site (iPBS) amplification. In total, 941 polymorphic amplified fragments were obtained using 12 ISSR, 12 SSR, ten AFLP, 19 iPBS, and 15 S-SAP primers or primer pairs. Four of the above-described techniques (except for SSRs with the primer pairs used in this study) were able to distinguish between the sports and their parental cultivar. The most effective technique to distinguish between the genotypes analysed was S-SAP, which detects variations in DNA regions flanking retrotransposon insertion sites.The combined use of ISSR,AFLP, iPBS, and S-SAP markers identified and distinguished all of the sports tested.     

Harvest parameters to improve the storability of high quality ‘Reinette du Canada’ apple

Summary

‘Reinette du Canada’ apple (Malus domestica Borkh.), which has been declared a Protected Designation of Origin (PDO) product throughout the European Community, is severely affected by bitter pit during storage, but increasing demand for organic produce requires chemical post-harvest treatments to be replaced with organic practices. The objective of this work was to determine the optimum stage of fruit maturity to improve the storability of high quality ‘Reinette du Canada’ apple during storage without applying post-harvest treatments. Seasonal conditions influenced fruit quality and the occurrence of storage disorders in ‘Reinette du Canada’. Harvest maturity also influenced the quality of ‘Reinette du Canada’ apple during storage, with later harvests reducing the development of bitter pit and increasing the soluble solids content:titratable acidity (SSC:TA) ratio. This study suggests that the SSC:TA ratio was a useful parameter for determining the optimum harvest maturity for high acidity, ‘Reinette du Canada’ apple. High quality fruit were obtained after long-term storage when the SSC:TA ratio exceeded 11.0, and fruit firmness at harvest exceeded 90 N. If these thresholds were not achieved, the quality of harvested ‘Reinette du Canada’ apples may not be sufficient to market fruit with the PDO designation. The results of our study also suggested that skin hue angle (hº) measurements may provide a quick and useful index that could replace fruit firmness measurements in the future.     

Thinning apples via shading: an appraisal under field conditions

Summary

Fruit development and the fate of fruits was followed on ‘Imperial Gala’ apple (Malus × domestica Borkh.) trees thinned chemically or using shading. The percentage fruit drop, fruit growth rates using callipers and electronic gauges, and whole tree gas exchange rates were measured before, during, and after covering with a shading cloth that blocked 90% of solar radiation, applied for 1 week starting 30 d after full bloom (DAFB). While fruits were thinned to similar crop loads and all reached a similar size at harvest, their growth patterns reflected the treatments applied, with fruit growth slowing down and fruit dropping sooner in chemically-thinned trees which were treated earlier, than in the shading treatment, which took place later. Daily fruit growth patterns changed and were greatly reduced under shading, as were the net carbon exchange rates (NCER), both in total per tree and per unit of light intercepted by the trees. Regression analysis of the NCER per unit of light intercepted revealed no difference between the two treatments before and after shading, but a significantly lower relationship during shading. This work supports the hypothesis that C-starvation may induce fruit abscission at approx. 30 DAFB. Fruit growth patterns, and their changes during shading, were consistent with this hypothesis. This method of thinning, may be of interest to reduce the use of chemicals, and in organic fruit growing. However, before it can be adopted, a method to estimate the length of the period of shading is required.     

不同品种苹果幼果期冻害比较及补救措施

以豫西地区果园5个不同苹果品种幼果为材料,比较果实的受冻情况。结果表明,不同品种间幼果的受冻程度存在明显差异,‘金冠’和‘瑞雪’受冻程度较轻,分别为37.5%和38.5%,‘嘎拉’受冻率次之,为61.0%,而‘富士’和‘红星’的受冻比例最大,达到78.0%和77.5%。同时,提出相关冻害补救措施,为苹果冻害防治提供技术参考。     

早熟苹果新品种‘华丹’

‘华丹’是以‘美八’与‘麦艳’杂交育成的早熟苹果新品种。果实近圆形,高桩,平均单果质量160 g;果实底色黄白,果面着鲜红色。果肉白色,肉质中细、松脆,风味酸甜,品质优良。果肉硬度6.3 kg · cm-2,可溶性固形物含量12.3%,可滴定酸含量0.49%。郑州地区果实7月初成熟,果实发育期80 ~ 85 d。盛果期产量可达45.0 t · hm-2。田间表现抗苹果枝干轮纹病和腐烂病,适宜在‘嘎拉’苹果种植区发展。     

The effect of selective picking on the harvest fruit size distribution of ‘Royal Gala‘ apples

Summary

The size distribution of fruit on an apple tree on any potential harvest date can be estimated by measuring a sample of fruit early in the season and projecting forward in time using standard growth curves. However, ‘Royal Gala‘ apples grown in New Zealand are picked in multiple harvests spread over about three weeks, so the overall size distribution of harvested fruit can differ significantly from that on any single harvest date. On each havest date, fruit are selected for picking according to background colour, not size. By measuring the sizes at harvest of all fruit on selected limbs of eight ‘Royal Gala‘ trees in Hawkes Bay, New Zealand, we show that the relationship between the size distributions of picked and unpicked fruit can be described by a single-parameter model. Although size is not used directly in fruit selection, we find that larger fruit still tend to be picked first, with the odds of a fruit being selected approximately doubling for each 20.g increase in size. The mean harvest fruit size is largest for the first harvest, and decreases in later harvests, despite ongoing fruit growth. Because the smaller fruit left on the trees continue to grow, the standard deviation of the overall fruit size distribution from multiple harvests can be expected to be significantly smaller than it would have been had all fruit been harvested on the same date. The model is tested using size and background colour measurements collected on tagged fruit at three sites in two years, including a range of shading and crop load treatments. For most combinations of site, year and treatment, the model predicted the size distribution of picked fruit well, but in a few cases a different parameter value was need to obtain a good fit.     

Effects of grafting height of MM106 rootstock on growth,lateral shoot formation and yield in apple trees

Summary

Between 2004 and 2008, the effects of different grafting heights on sylleptic shoots were tested in the apple (Malus domestica Borkh.) cultivars ‘Granny Smith’ and ‘Gloster’, and cumulative fruit yields were evaluated. MM106 apple rootstocks were grafted 10, 20, 40, or 60 cm above soil level in August 2004. The results showed that an increased grafting height significantly decreased tree height in both cultivars. The tallest and shortest trees were observed at grafting heights of 10 cm (153.0 and 170.0 cm) and 60 cm (141.3 and 143.5 cm) in ‘Granny Smith’ and ‘Gloster’, respectively.Among the various grafting heights tested, 60 cm in ‘Granny Smith’ and 20 cm in ‘Gloster’ gave the largest stem diameters (17.6 mm and 16.8 mm, respectively). The number of lateral shoots increased significantly with increased grafting height in both cultivars. The largest numbers of lateral shoots in ‘Granny Smith’ (10.75) and ‘Gloster’ (2.00) were obtained from a grafting height of 60 cm, while 2.55 and zero lateral shoots occurred at 10 cm grafting height in ‘Granny Smith’ and ‘Gloster’, respectively. Shoot lengths decreased significantly by increasing the grafting height. Grafting heights of 10 cm and 60 cm resulted in the tallest and shortest shoots in both cultivars. Cumulative fruit yields were significantly affected by grafting height in both cultivars. The highest yield was found for a 60 cm grafting height in both ‘Granny Smith’ (11.295 kg tree^–1) and ‘Gloster’ (4.818 kg tree^–1).The results of this study suggest that grafting heights of 40 cm and 60 cm have the potential to promote branching and early bearing for apple fruit production in sustainable and organic agricultural systems.     

Ultrastructure of epicuticular wax aggregates during fruit development in apple (Malus domestica Borkh.)

Summary

Development of wax platelets on the surface of ‘Delicious’ (Malus domestica Borkh.) apple fruit was investigated throughout the growing season using field emission scanning electron microscopy. At 5,000 and greater, wax crystalline structures appeared to be composed of microtubules (MT), aggregates of individual MT to form single platelets, and of one or more platelets. The thickness of a single wax platelet ranged from 116–128 nm, approximately the diameter of a single MT; whereas multiple-platelet aggregates ranged in thickness from 307–428 nm. Individual platelets, multiple platelet aggregates and MT were visible throughout fruit development, as well as on different apple cultivars. Examination of the cuticle from young fruitlets (receptacle diameter = 3 mm) of ‘Chinese Crabapple’ (M. hupehensis Rehd.), which develops without trichomes, best demonstrated early platelet formation. A model for wax platelet and cuticle development on apple is proposed based on these data.