Cryopreservation of winter-dormant apple: III Bud water status and survival after cooling to -30°c and during recovery from cryopreservation

Abstract In a continuing study to improve the efficiency of dormant bud cryopreservation for tissues hardened in maritime climates, the water status of dormant buds was monitored between -4°C and recovery from liquid nitrogen (LN). Measurement of water content, simple thermal analysis and differential scanning calorimetry were employed. Buds did not lose water during cooling to, or holding at -30°C indicating that cryodehydration and/or other adaptive responses contributed during this essential step. A bud exotherm that was an artefact of warming was detected due to necessary handling at -4°C before cooling to -30°C. There were no significant differences between cultivars with respect to water status at -30°C or immediately upon rewarming from LN despite significant differences in post-LN survival. Buds rehydrated in 5 days, but up to 14 days may be needed for recovery for some cultivars. In some instances buds could be grafted without rehydration, taking up water across the early graft union.

Cryopreservation of winter-dormant apple buds: II - tissue water status after desiccation at -4°c and before further cooling

Abstract The established protocol for the cryopreservation of winter-dormant Malus buds requires that stem explants, containing a single, dormant bud are desiccated at -4°C, for up to 14 days, to reduce their water content to 25-30% of fresh weight. Using three apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90°C) of intact buds indicated that bud tissues of the cultivar with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature Keywords: Malus, cryopreservation, dormant bud, dehydration

Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array

Background A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNPbased linkage map of an apple rootstock progeny. Results Of the 7,867 Malus SNP markers on the array, 1,823 (23.2 %) were heterozygous in one of the two parents of the progeny, 1,007 (12.8 %) were heterozygous in both parental genotypes, whilst just 2.8 % of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the ‘Golden Delicious’ genome sequence. A total of 311 markers (13.7 % of all mapped markers) mapped to positions that conflicted with their predicted positions on the ‘Golden Delicious’ pseudo-chromosomes, indicating the presence of paralogous genomic regions or misassignments of genome sequence contigs during the assembly and anchoring of the genome sequence. Conclusions We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the ‘Golden Delicious’ reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.

The Glu298Asp single nucleotide polymorphism in the endothelial nitric oxide synthase gene differentially effects the vascular response to acute consumption of fruit and vegetable puree-based drinks

Scope Diets low in fruits and vegetables (FV) are responsible for 2.7 million deaths from cardiovascular diseases (CVD) and certain cancers annually. Many FV and their juices contain flavonoids, some of which increase endothelial nitric oxide synthase (eNOS) activity. A single nucleotide polymorphism in the eNOS gene, where thymine (T) replaces guanine (G) at position 894 predicting substitution of glutamate for aspartate at codon 298 (Glu298Asp), has been associated with increased CVD risk due to effects on nitric oxide synthesis and subsequently vascular reactivity. Individuals can be homozygous for guanine (GG), thymine (TT) or heterozygous (GT). Methods and results We investigated the effects of acute ingestion of a FV-puree-based-drink (FVPD) on vasodilation and antioxidant status in subjects retrospectively genotyped for this polymorphism. Healthy volunteers (n = 24; 11 GG, 11 GT, 2 TT) aged 30–70 were recruited to a randomized, controlled, crossover, acute study. We showed that acute consumption of 400 mL FVPD differentially affected individuals depending on their genotype. There was a significant genotype interaction for endothelium-dependent vasodilation measured by laser Doppler imaging with iontophoresis (P < 0.05) and ex vivo low-density lipoproteins (LDL) oxidation (P = 0.002). GG subjects had increased endothelium-dependent vasodilation 180 min (P = 0.028) and reduced ex vivo LDL oxidation (P = 0.013) after 60 min after FVPD compared with control, no differences were observed in GT subjects. Conclusion eNOS Glu298Asp genotype differentially affects vasodilation and ex vivo LDL oxidation after consumption of FV in the form of a puree-based drink.

Effects of chronic consumption of fruit and vegetable puree-based drinks on vasodilation, plasma oxidative stability and antioxidant status

Background: Fruit and vegetable-rich diets are associated with a reduced cardiovascular disease (CVD) risk. This protective effect may be a result of the phytochemicals present within fruits and vegetables (F&V). However, there can be considerable variation in the content of phytochemical composition of whole F&V depending on growing location, cultivar, season and agricultural practices, etc. Therefore, the present study investigated the effects of consuming fruits and vegetables as puree-based drinks (FVPD) daily on vasodilation, phytochemical bioavailability, antioxidant status and other CVD risk factors. FVPD was chosen to provide a standardised source of F&V material that could be delivered from the same batch to all subjects during each treatment arm of the study. Methods: Thirty-nine subjects completed the randomised, controlled, cross-over dietary intervention. Subjects were randomised to consume 200 mL of FVPD (or fruit-flavoured control), daily for 6 weeks with an 8-week washout period between treatments. Dietary intake was measured using two 5-day diet records during each cross-over arm of the study. Blood and urine samples were collected before and after each intervention and vasodilation assessed in 19 subjects using laser Doppler imaging with iontophoresis. Results: FVPD significantly increased dietary vitamin C and carotenoids (P < 0.001), and concomitantly increased plasma α- and β-carotene (P < 0.001) with a near-significant increase in endothelium-dependent vasodilation (P = 0.060). Conclusions: Overall, the findings obtained in the present study showed that FVPD were a useful vehicle to increase fruit and vegetable intake, significantly increasing dietary and plasma phytochemical concentrations with a trend towards increased endothelium-dependent vasodilation.

Effects of acute consumption of a fruit and vegetable puree-based drink on vasodilation and oxidative status

Epidemiological studies indicate that diets rich in fruits and vegetables (F&V) are protective against cardiovascular diseases (CVD). Pureed F&V products retain many beneficial components, including flavonoids, carotenoids, vitamin C and dietary fibres. This study aimed to establish the physiological effects of acute ingestion of F&V puree-based drink (FVPD) on vasodilation, antioxidant status, phytochemical bioavailability and other CVD risk factors. 24 Subjects, aged 30-70 years, completed the randomised, single-blind, controlled, crossover test meal study. Subjects consumed 400 ml FVPD, or fruit-flavoured sugar-matched control, after following a low-flavonoid diet for 5 days. Blood and urine samples were collected throughout the study day and vascular reactivity was assessed at 90-minute intervals using laser Doppler iontophoresis (LDI). FVPD significantly increased plasma vitamin C (P=0.002) and total nitrate/nitrite (P=0.001) concentrations. There was a near significant time by treatment effect on ex vivo LDL oxidation (P=0.068), with a longer lag phase after consuming FVPD. During the 6 hours after juice consumption the antioxidant capacity of plasma increased significantly (P=0.003) and there was a simultaneous increase in plasma and urinary phenolic metabolites (P<0.05). There were significantly lower glucose and insulin peaks after ingestion of FVPD compared with control (P=0.019 and P=0.003) and a trend towards increased endothelium-dependent vasodilation following FVPD consumption (P=0.061). Overall, FVPD consumption significantly increased plasma vitamin C and total nitrate/nitrite concentrations, with a trend towards increased endothelium-dependent vasodilation. Pureed F&V products are useful vehicles for increasing micronutrient status, plasma antioxidant capacity and in vivo NO generation, which may contribute to CVD risk reduction.

Survival of freeze dried Lactobacillus plantarum in instant fruit powders and reconstituted fruit juices

The aim of this study was to investigate the survival of freeze dried Lactobacillus plantarum cells mixed with several freeze dried instant fruit powders (strawberry, pomegranate, blackcurrant and cranberry) during storage for 12 months as well as after reconstitution with water each month. Inulin and gum arabic were also added to the instant fruit powders at two levels (10% and 20% w/w of dry weight) to improve the cell survival and functional properties of the product. The best cell survival over the 12 months of storage was observed for the blackcurrant powder (almost no decrease) followed by strawberry (~ 0.3 log decrease), pomegranate (~ 0.9 log decrease), whereas the worst survival was obtained in cranberry powder (~ 4.5 logs). To explain these results multiple regression analysis was conducted with the log decrease [log10N0 month − log10N12 months] as the dependent variable and water activity, pH, citric acid, dietary fibre and total phenol as the independent variables. The results indicated that among all the examined factors, the [log10N0 month − log10N12 months] depended only on the water activity (P < 0.05). Inulin and gum arabic demonstrated a substantial protective effect on cell survival (1–1.5 log) in the case of cranberry, which was likely due to a physical interaction between the cells and the carbohydrates. After reconstituting the dried fruit powders at room temperature and measuring cell viability for up to 4 h, it was shown that in the case of strawberry juice there was no decrease, and very little in the case of pomegranate and blackcurrant juices (< 0.5 log). On the other hand, a significant decrease was observed for cranberry juice (P < 0.05), which increased as the storage time of the dried cranberry powder increased, indicating that the cells became more susceptible with prolonged storage. Multiple regression analysis indicated that the main factors influencing cell survival were water activity and pH, while citric acid, dietary fibre and total phenol did not have an effect. Furthermore, inulin and gum arabic addition did not have a significant (P > 0.05) effect upon reconstitution of the dried fruit powder. This study showed that instant juice powders are very good carriers of probiotic cells and constitute good alternatives to highly acidic fruit juices.

Wild pollinators enhance fruit set of crops regardless of honey bee abundance

Diversity and abundance of wild-insect pollinators have declined in many agricultural landscapes. Whether such declines reduce crop yields, or are mitigated by managed pollinators such as honey bees, is unclear. Here, we show universally positive associations of fruit set with wild-insect visits to flowers in 41 crop systems worldwide, and thus clearly demonstrate their agricultural value. In contrast, fruit set increased significantly with visitation by honey bees in only 14% of the systems surveyed. Overall, wild insects pollinated crops more effectively, because increase in their visitation enhanced fruit set by twice as much as an equivalent increase in honey bee visitation. Further, visitation by wild insects and honey bees promoted fruit set independently, so high abundance of managed honey bees supplemented, rather than substituted for, pollination by wild insects. Our results suggest that new practices for integrated management of both honey bees and diverse wild-insect assemblages will enhance global crop yields.

Effect of cultivar type and ripening on the polyphenol content of date palm fruit

Date palm (Pheonix dactylifera) fruit contains an array of polyphenols, although how these levels alter with cultivar type and fruit ripening is unclear. Utilizing HPLC and LC-ESI-MS/MS, this study define and quantify an array of hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids in three common cultivars of dates (Ajwa, Barni, and Khalas) at the main ripening stages (kimri, khalal, rutab, and tamr). Polyphenols were at highest concentration at earlier stages of ripening, with concentrations reducing with ripening. The khalal stage of the Ajwa cultivar contained significantly higher (P < 0.001) levels of polyphenols than measured in the Barni and Khalas dates at the same degree of ripening. Furthermore, the Ajwa cultivar was the only one to contain significant quantities of anthocyanidins, in particular at the khalal stage. These data suggest dates are a significant source of polyphenols, especially if the earlier edible ripening stages are consumed or utilized as food ingredients.

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing.

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.

Influence of encapsulation and coating materials on the survival of Lactobacillus plantarum and Bifidobacterium longum in fruit juices

The aim of this work was to compare alginate and pectin beads for improving the survival of Lactobacillus plantarum and Bifidobacterium longum during storage in pomegranate and cranberry juice, and to evaluate the influence of various coating materials, including chitosan, gelatin and glucomannan on cell survival and on the size and hardness of the beads. In pomegranate juice, free cells of L. plantarum died within 4 weeks of storage and those of B. longum within 1 week; in cranberry juice both types of cells died within one week. Encapsulation within either alginate or pectin beads improved cell survival considerably, but coating of the beads with chitosan or gelatin improved it even further; coating with glucomannan did not have any positive effect. The double gelatin coated pectin beads gave the highest protection among all types of beads, as a final concentration of approximately 108 CFU/mL and 106 CFU/mL for both L. plantarum and B. longum was obtained after 6 weeks of storage in pomegranate and cranberry juice, respectively. The good protection could be attributed to the very strong interaction between the two polymers, as measured by turbidity experiments, leading to the formation of a polyelectrolyte complex. It was also shown that the coating was able to inhibit the penetration of gallic acid within the beads, which was used in this study as a model phenolic compound with antimicrobial activity; this is a likely mechanism through which the beads were able to protect the cells from the antimicrobial activity of phenolic compounds present in both types of juices. Despite their good protective effect, the pectin beads were considerably softer than the alginate beads, an issue that should be addressed in order to increase their mechanical stability.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria X ananassa Duch.) fruit, a non-climacteric tissue

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

Polyphenols, glucosinolates, dietary fibre and colon cancer: Understanding the potential of specific types of fruit and vegetables to reduce bowel cancer progression

Colorectal cancer is the third most prevalent cancer worldwide and the most common diet-related cancer, influenced by diets rich in red meat, low in plant foods and high in saturated fats. Observational studies have shown that fruit and vegetable intake may reduce colorectal cancer risks, although the precise bioactive components remain unclear. This review will outline the evidence for the role of polyphenols, glucosinolates and fibres against cancer progression in the gastrointestinal tract. Those bioactive compounds are considered protective agents against colon cancer, with evidence taken from epidemiological, human clinical, animal and in vitro studies. Various mechanisms of action have been postulated, such as the potential of polyphenols and glucosinolates to inhibit cancer cell growth and the actions of insoluble fibres as prebiotics and the evidence for these actions are detailed within. In addition, recent evidence suggests that polyphenols also have the potential to shift the gut ecology in a beneficial manner. Such actions of both fibre and polyphenols in the gastrointestinal tract and through interaction with gut epithelial cells may act in an additive manner to help explain why certain fruits and vegetables, but not all, act to differing extents to inhibit cancer incidence and progression. Indeed, a focus on the individual actions of such fruit and vegetable components, in particular polyphenols, glucosinolates and fibres is necessary to help explain which components are active in reducing gastrointestinal cancer risk.

Exposure to foods’ non-taste sensory properties: a nursery intervention to increase children’s willingness to try fruit and vegetables

Activities that engage young children with the sensory properties of foods are popular with nursery schools, despite the lack of evidence for their efficacy in increasing children's consumption of healthy foods. This study provides the first empirical exploration of the effectiveness of a non-taste sensory activity program in a nursery school setting. Ninety-two children aged between 12 and 36 months were allocated to either an intervention group, who took part in looking, listening, feeling and smelling activities with unusual fruits and vegetables every day for four weeks, or to a non-intervention control group. In a subsequent mealtime taste test, children touched and tasted more of the vegetables to which they had been familiarized in their playtime activities than of a matched set of non-exposed foods. The results demonstrate that hands-on activities with unfamiliar fruits and vegetables can enhance children’s willingness to taste these foods, and confirm the potential for such activities to support healthy eating initiatives.

Characterization of the lipoxygenase (LOX) gene family in the Chinese white pear (Pyrus bretschneideri) and comparison with other members of the Rosaceae

Background Lipoxygenases (LOXs), a type of non-haem iron-containing dioxygenase, are ubiquitous enzymes in plants and participate in the formation of fruit aroma which is a very important aspect of fruit quality. Amongst the various aroma volatiles, saturated and unsaturated alcohols and aldehydes provide the characteristic aroma of the fruit. These compounds are formed from unsaturated fatty acids through oxidation, pyrolysis and reduction steps. This biosynthetic pathway involves at least four enzymes, including LOX, the enzyme responsible for lipid oxidation. Although some studies have been conducted on the LOX gene family in several species including Arabidopsis, soybean, cucumber and apple, there is no information from pear; and the evolutionary history of this gene family in the Rosaceae is still not resolved. Results In this study we identified 107 LOX homologous genes from five Rosaceous species (Pyrus bretschneideri, Malus × domestica, Fragaria vesca, Prunus mume and Prunus persica); 23 of these sequences were from pear. By using structure analysis, phylogenic analysis and collinearity analysis, we identified variation in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Expression of certain pear LOX genes during fruit development was verified by analysis of transcriptome data. Conclusions 23 LOX genes were identified in pear and these genes were found to have undergone a duplication 30–45 MYA; most of these 23 genes are functional. Specific gene duplication was found on chromosome4 in the pear genome. Useful information was provided for future research on the evolutionary history and transgenic research on LOX genes.