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.     

Anatomical and morphological study of apple (Malus × domesticaBorkh.) flower buds growing under inadequate winter chilling

Summary

Some apples cultivars produce low yields when grown in regions with inadequate winter chilling. Their unsatisfactory development is attributed to the lack of climatic adaptation which causes some abnormalities in bud differentiation. The development of reproductive spurs is erratic, leading to vegetative shoots, and the flower index is very low. The purpose of this work is to understand the flower differentiation problem. An assessment was made through morphological and histological studies, also an analysis of climatic data was performed in an attempt to identify the responsible factors. The number of chilling hours recorded was about 695. Defoliation was delayed and happened during the second week of January. Bud break was advanced by 10 d in comparison with the average period. The spurs density (12 and 23 spurs per m of twigs) was similar to the values observed in normal situation. The buds carried by these spurs evolved into vegetative shoots for all variety × rootstock combination used in this study. The average of this transformation was 47 and 50% for ‘Golden Delicious’ and ‘Starking Delicious’, respectively. A flowering index obtained was very low (1.3 kg per tree). Anatomical observations carried out on buds collected in October showed that differentiation was undertaken and the floral primordium was already formed with some abnormalities in flower development in later stage. At anthesis, internal structures of the buds showed primordia disorganized. Reproductive organs presented pistil abortion with low microsporogenesis. Xylem vessel elements were not observed at the base of the bud and vascular connection was not established. This problem in flower development occurred at this stage which was affected by external factors. The winter was characterized by periods of high temperatures which affect negatively the accumulation of chilling units. The mode of action of temperature, notably of chilling on the formation of flowering organs and vessels, remains to be determined.     

Comparison of the compatibility and metaxenia effects of several dessert apple and ornamental Malus cultivars with Cox’s orange pippin

Flowers of apple cv Cox’s Orange Pippin were hand-pollinated with diluted or undiluted pollen of six dessert and five ornamental Malus cultivars. Subsequent examinations of pollen tube growth, fruit set and seed numbers showed that all were compatible with Cox, though some differences were found. Pollen of four of the five ornamental Malus cvs, viz. Golden Hornet, Hillieri, Winter Gold and Aldenhamensis, was as compatible with Cox as that of the dessert cultivars. No metaxenia effects on size, weight, colour, russet, acidity or sugar content were seen in the pollinated Cox fruit.     

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.     

The effect of boron upon leaf development and growth of the apple cultivar ‘Cox’s Orange Pippin’

One-year-old ‘Cox’s Orange Pippin’ trees planted in pots in sand culture in the spring of 1970 were (during the growing seasons of 1970–72) watered with nutrient solutions containing 0, 0.12, 0.23, 1.2, 3.5 or 7.0 mg boron per litre.

With increasing boron supply, the boron concentration of the leaves increased, the growth of current year’s shoots and to some extent of other plant parts increased, and summer leaf-fall and the occurrence of necrotic leaf spots (‘Cox spots’) decreased. The percentage of healthy leaves was greatest when boron in leaf dry matter was about 30 ppm.

In 1972 the trees with highest boron supply suffered from incipient boron toxicity and increased incidence of ‘Cox disease’ flowering was delayed, and the percentage of dead buds was increased. In the latter part of the summer the leaves curled upwards, turned purple and became brittle.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

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.     

Effects of genotype and environment on total anti-oxidant capacity and the content of sugars and acids in strawberries (Fragaria × ananassa Duch.)

Summary

Total anti-oxidant capacity (TAC) and the contents of mono- and disaccharides, and organic acids were determined in strawberry fruit from ten genotypes sampled from eight experimental sites in Norway in 2002 and 2003. The difference between genotypes was significant for all recorded traits, and it appeared possible to select for all traits in breeding programmes. On average, the Norwegian bred cultivar ‘Carmen’ had a TAC of 30.07 mmol kg^–1 fresh weight (FW), compared to 23.16 mmol kg^–1 FW in the standard cultivar ‘Korona’. TAC was negatively correlated with fruit size, rainfall and leaf surface humidity, but was positively correlated with the minimum temperature on the day prior to sampling. Mono- and di-saccharide contents were negatively correlated with both minimum and maximum temperatures, and with wind velocity. It was confirmed that the sugars:organic acids ratio was inversely related to the maximum temperature on the day before harvest, which supports anecdotal claims that strawberries grown in northern areas have, on average, better flavour.     

The Relationship Between Growth Vigour of Rootstock and Phenolic Contents in Apple (Malus × domestica)

The aim of the study is to determine the effect of different growth vigorous rootstocks on phenolic compounds in leaves of apple. For this purpose it was used the leaves of cultivar ‘Red Chief’ grafted on dwarf (M9), semi-dwarf (M26) and semi-vigorous (MM106) rootstocks. During mid-July, the leaf samples were taken from the middle part of annual shoots. Phenolics of the leaves were determined by HPLC analysis. While significant differences among the rootstocks for p-hydroxybenzoic acid, eriodictyol, ferulic acid and p-coumaric acid were detected, these differences were insignificant for gallic acid and quercetin. It was shown that semi-vigorous rootstocks (MM106) had higher phenolic contents in total than the other two dwarf rootstocks. In addition, apigenin-7-glucoside, chlorogenic acid, caffeic acid, rosmarinic acid, epicatechin, syringic acid, catechin, rutin, resveratrol, hesperidin, naringenin, luteolin, apigenin and acacetin could not to be detected. Data showed that there is the relationship between growth vigour and phenolic contents of apple leaves. Especially, p-hydroxy benzoic acid and p-coumaric acid contents were higher in semi-vigorous rootstock than in dwarf rootstock.     

Analysis of differentially expressed proteins in the pollen of Malus spectabilis (crabapples) and Malus domestica (‘Gala’ apples) and their role in pollination

Our objective was to study the differentially expressed proteins (DEP) in various Malus spectabilis (crabapple) varieties (M. ‘Snowdrift’, M. ‘Hongling’ and M. ‘Hongjin’) in relation to Malus domestica (‘Gala’) and their role in pollination. Our method used a two-dimensional electrophoresis (2-DE) to analyse the differential proteins in the pollen of several crabapples. The 2-DE apples combined with the tandem mass spectrometry (MS-MS) and protein database retrieval helped us to identify the nature and function of DEPs in ‘Gala’ apples and crabapples. We identified 1195 proteins through 2-DE. Among these, six DEPs, namely chloroplast ferritin, Actin, Beta-fructofuranosidase, vacuolarH+-ATPase catalytic subunit, Full = Phosphoglucomutase, and Cytochrome b were identified by MS-MS. This study identified six DEPs among the pollen from the ‘Snowdrift’ crabapple, ‘Hongling’ crabapple, ‘Hongjin’ crabapple, and ‘Gala’ apples. The DEPs included metabolism related proteins, stress/regulatory proteins, and proteins involved in signal transduction.     

Pre-harvest drop control of ‘Red Delicious’ and ‘Jonathan’ apple (Malus domestica) as affected by the synthetic auxin 3,5,6-TPA

Summary

‘Red Delicious’, the main apple cultivar grown in Israel, and ‘Jonathan’, grown on a smaller scale, are prone to substantial pre-harvest drop. Spraying both cultivars with the synthetic auxin 3,5,6-trichloro-2-pyridyl-oxyacetic acid (3,5,6-TPA) reduced pre-harvest fruit drop and increased yield. Application of 40 mg l^–1 3,5,6-TPA 2 weeks before anticipated harvest (WBH), or 30 mg l^–1 3,5,6-TPA at 4 and 2 WBH gave the best results, and were as effective as 2,4,5-trichloro-phenoxypropionic acid (2,4,5-TP) which was the commercial product commonly in use until being withdrawn. There were no negative effects on fruit quality at harvest, or during 8 months of storage. Therefore 3,5,6-TPA can be recommended for routine use on ‘Red Delicious’ and ‘Jonathan’ apples in Israel.     

Regeneration from in-vitro leaves of ‘Conference’ and other pear cultivars (Pyrus communis L.)

Summary

Regeneration of shoots from in-vitro grown leaf tissues of the pear cv. Conference was achieved at rates of up to 40%. The highest regeneration percentage was achieved using a phytohormone combination of benzylamino purine (5 mg 1^?1) and 1-naphthalene acetic acid (1.0 mg 1^?1) on a basal medium of Murashige and Skoog, to which had been added 200 mg I^?1 of the antibiotic cefotaxime. The percentage regeneration was reduced when a higher concentration of cefotaxime (400 mg 1^?1) was used. Cefotaxime had little beneficial effect on regeneration at a lower concentration of 1-naphthalene acetic acid (0.5 mg l^?1). Regenerated shoots were easily micropropagated, rooted and transplanted to soil. To date, the plants have a true-to-type phenotypic appearance. Regeneration was also achieved with other cultivars—‘Abbé Fetel’, ‘Doyenne d’Hiver’, ‘Doyenne du Comice’ and ‘Passé Crassane’—but percentage regeneration was lower and, apart from ‘Passé Crassane’, the regenerated shoots were weak and could not be subcultured.     

In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, ‘Fertility’

Summary

Polyploid plantlets, including triploid, tetraploid, and mixoploid, were induced from the European pear (Pyrus communis L.) cultivar ‘Fertility’ by in vitro colchicine treatment of leaf explants. The leaf explants were incubated in 0.4% (w/v) colchicine for 24, 48, or 72 h, then transferred to adventitious shoot-induction medium. Regenerated shoots were pre-selected according to their morphological characteristics when compared to control shoots from untreated shoot proliferation cultures. Shoots with putative polyploid morphological characteristics were maintained and proliferated. The ploidy levels of all putative polyploid individuals were analysed by flow cytometry and identified by chromosome counts of shoot tip tissue squashes. Polyploid shoots were rooted, and the resulting plantlets were transferred to the field. Polyploid plantlets had a higher specific leaf mass and larger stomata than those of diploid plantlets.     

The RÔLe of Secondary Infections in the Spread of Apple Mildew (Podosphaera Leucotricha (Ell. and Ev.) Salm.)

Firmness measurements made on tomato fruit at various degrees of ripeness by means of a machine recently described have been compared with those produced by an established instrument. Highly significant correlations were obtained between results from the two types of apparatus. Because of its greater ease of operation the newer instrument was used to compare the firmness of six varieties of tomato fruit, all judged to be in a uniform condition known as the “ commercial picking stage ”. The polygalacturonase activity in the same tomato samples was also measured, and a highly significant positive correlation established between the activities of the enzyme and the compression of the different varieties of fruit under a standard load. These results strongly suggest that pectolytic enzyme activity is one of the predominant factors governing the firmness of different varieties of tomato fruit.     

The influence of planting date,sucker selection and density on yield and crop-timing of bananas (cultivar ‘Williams’) in the Eastern Transvaal

In the subtropical banana-growing areas of South Africa, there is a pronounced and consistent tendency for fruit to be oversupplied in the spring (September–November) and undersupplied in the autumn (March–May). Under identical soil, planting material and general management conditions, a crop-timing trial with ‘Williams’ banana was established at Burgershall Research Station, Eastern Transvaal, to compare the effects of planting date (September, December, March), time of first sucker selection (5 and 10 months after planting) and density (1666 and 1250 plants ha⁻¹) on yield and harvest season over 3 crop cycles.Cumulating the yield/ha/annum for the plant crop and first ratoon cycles, there was a small but significant decrease (4%) as planting date was delayed from September to December, and a larger significant reduction in yield (18%) with delay from December to March planting. While cumulative yield/ha/annum for plant crop plus first ratoon increased significantly (19%) at the higher density of 1666 plants ha⁻¹, no differences occurred as a result of sucker selection treatment.March-planted bananas were harvested during the undesirable spring period, and this effect was largely carried over into the first ratoon and second ratoon cycles. December planting was optimal and September planting intermediate from a crop-timing viewpoint. Cumulating all 3 crop cycles, 50% of the total bunch harvest could be timed during the autumn from December planting at a density of 1666 plants ha⁻¹. From September planting, the proportion of autumn-harvested fruit could also be increased by delaying the selection of the first ratoon sucker until at least 10 months after planting. Results demonstrated the in-field potential for overcoming the natural banana shortage in South Africa during autumn.     

Influence of soil hydrothermal environment,irrigation regime,and different mulches on the growth and fruit quality of strawberry (Fragaria × ananassa L.) plants in a sub-temperate climate

Summary

The present study was undertaken over two consecutive years under sub-temperate climatic conditions in the mid-hill region of Himachal Pradesh (30°52'N, 77°11'E; 1,175 m asl) on loamy sand Inceptisols. The aim was to investigate the effects of irrigation and mulch material on the growth, flowering, fruiting behaviour, relative leaf water content (RLWC), yield, and quality of strawberry (Fragaria × ananassa L. ‘Chandler’). The drip irrigation treatments included irrigation with 100, 80, or 60% (coded 1.0, 0.8, and 0.6 V) of the total water requirement. Both mulches increased the minimum soil temperature to a depth of 5 cm. The drip and surface irrigation treatments raised the minimum soil temperature by 3.0º – 5.4ºC, and lowered the maximum temperature by 2.2º – 5.8ºC compared to the rainfed control. Hay mulch was more effective in raising the minimum temperature and lowering the maximum soil temperature than black polyethylene mulch. Moisture conservation increased by 2.8 – 12.8% under the black polyethylene mulch compared to the no-mulch treatment. Drip and surface irrigation methods, as well as mulching, were found to be effective for enhancing the growth, irrigation water use efficiency (IWUE), fruit yield, and quality of strawberry plants. However, the number of crowns per plant, the percentage berry set, the RLWC, root length density (RLD), and fruit yield were highest under treatment M3I3 [i.e., black polyethylene mulch plus drip irrigation (1.0 V)] by 565.5%, 94.5%, 32.8%, 394.5%, and 549.6%,respectively, compared to the no mulch plus rainfed control. The maximum IWUE values for plant biomass [1.39 metric tonnes (MT) ha^–1 m^–1] and fruit yield (2.79 MT ha^–1 m^–1) were recorded under treatment M3I5 [i.e., black polyethylene mulch plus drip irrigation (0.6 V)]; whereas, the lowest values (0.39 and 0.68 MT ha^–1 m^–1, respectively) were observed using treatment M1I2 (i.e., without mulch, plus surface irrigation). Fruit size, weight, sugar content, and anthocyanin content increased significantly under treatment M2I3 [i.e., hay mulch, plus drip irrigation (1.0 V)] compared with all other treatments. Total soluble solids (TSS) contents and total acidity (TA) were highest under treatment M1I1 (i.e., no mulch, plus rainfed). A linear regression model could describe the variations in quality parameters of strawberry plants grown under sub-temperate climatic conditions. Root density was found to be the best indicator with which estimate fruit quality.     

Studies on the hawthorn (Crataegus azarolus L.): III. A potential rootstock for ‘Golden Delicious9 apple and ‘Williams’ pear

Seedlings of the hawthorn Crataegus azarolus L. were budded with ‘Williams’ pear and ‘Golden Delicious’ apple and followed for compatibility, growth control, precocity and suckering. Four years of observations indicated that the hawthorn is compatible with ‘Williams’ and ‘Golden Delicious’, conferring growth control and precocity; and producing suckers profusely. On the other hand, bud break in ‘Williams’ started 10 d earlier and gave longer shoots than ‘Golden Delicious’. Delayed symptoms of incompatibility and long-term reciprocal influences require further study.