The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of York and Vermont Spur Delicious apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K⁺, Mg⁺⁺ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K⁺, 1.5 and 3.0 mM Mg⁺⁺, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K⁺ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

Water relations,mineral nutrition,growth, and13C discrimination in two apple cultivars under daily episodes of high root-medium temperature

Although high soil temperatures can occur in apple orchards throughout the world, there is little information on their effect. This investigation was conducted to determine the influence of various durations of root exposure to 34 °C on the growth and physiology of the apple plant. Roots of Royal Gala and McIntosh cultivars were exposed to 34 °C for 0, 8, 16, and 24 hours/day for seven weeks. Royal Gala grown at the 24 hours/day treatment exhibited significant decreases in leaf, shoot, and root growth; chlorophyll concentration of the older leaves; transpiration; discrimination against¹³C in leaves; and an increase in leaf temperature. In McIntosh, root growth and chlorophyll concentration of leaves were not affected. For both cultivars compared to the control treatment, the continuous high temperature treatment resulted in lower levels of P, Mg, and Mn in leaves. Royal Gala at this treatment showed significantly higher values of foliar N and K and lower values of Ca, Fe, and Zn. For McIntosh the levels of Cu and B decreased significantly in this treatment as compared to the control treatment. We conclude that 34 °C in the root-zone does not stress these cultivars unless it persists throughout the day/night cycle.     

A new,efficient method using 8-hydroxy-quinolinol-sulfate for the initiation and establishment of tissue cultures of apple from adult material

Applying the new method for culture initiation 16 different cultivars of Malus domestica could be established in vitro from shoot tips of adult orchard trees. Actively growing shoot tips were cleaned and surface disinfested, dissected to 2–3 mm and placed on a modified MS-medium with 4.4 M BA. Explants were covered for 24 h with 200 l of a 0.1% solution of 8-hydroxy-quinolinol-sulfate (8-HQS) and transferred to a medium containging both auxin and cytokinin after 2 weeks. The application of 8-HQS induced a strong reduction of the infection rate and inhibited the browning of the explants and the media. After 7 days yields of 50–90% sterile explants could be obtained in comparison to 100% losses of untreated shoot tips. After 60 days variable rates of actively growing shoots could be observed, depending on the genotypes. The described method allows a successful establishment of fruit trees from adult orchard material on one hand by strongly reducing the browning, caused by the oxidation of polyphenolic compounds by polyphenoloxidases, and on the other hand 8-HQS can strongly increase the yield of explants without contamination, independently from the vegetation period and the phytosanitary state of the donor material.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - 8-HQS 8-hydroxy-quinolinol-sulfate     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

CO2-Enrichment and photosynthetic photon flux affect the growth of in vitro-cultured apple plantlets

Micro-cuttings (shoots with two small leaves) of cultivar M9 apple were cultured in-vitro for 40 d under CO₂-enriched and non-enriched (i.e., ambient air) conditions, and at a PPF of 40 or 100 μmol m^-2 s^-1 Afterward, shoot length, number of leaves, leaf area, chlorophyll content, shoot and root fresh weights, and % survival were recorded. Those plant-lets grown under CO₂- and PPF-enriched treatments were healthy and vigorous, and showed higher values for their growth parameters. In contrast, those grown without supplemental CO₂ or PPF often showed hyperhydricity. We also demonstrated that CO₂ enrichment and a relatively high PPF during in-vitro culture promoted normal photosynthesis and growth after ex-vitro transplantation.     

Biosynthesis of phenolic compounds and its regulation in apple

This paper summarises the information on the occurrence of phenolic compounds in apple Malus domestica leaves and fruits with special reference to their developmental changes and regulation of biosynthesis. Besides the ontogenetic variation in biosynthesis and accumulation, the stress-induced and pathogenesis-related changes are emphasised. Aspects of commercial importance are addressed, ranging from fruit colouration, through disease resistance, to the direct use of apple tissues, as raw material for the extraction of bioactive phenolic compounds.     

Factors that effect leaf regeneration efficiency in apple,and effect of antibiotics in morphogenesis

Several factors that affect the frequency of organogenesis in apple leaf explants were examined for the scion cultivars Empire, Freedom, Golden Delicious, Liberty, McIntosh, and Mutsu and for the rootstocks Malling 7A and Malling 26. The main factors affecting morphogenesis were BA concentration, basal medium, leaf explant origin and maturity, explant orientation, and photosynthetic photon flux. Depending on the genotype, optimal regeneration was obtained using either 22.2 or 31.1 M BA and the N6 basal medium, with the exception of Golden Delicious which regenerated better on MS medium. After 6 weeks, the average number of shoots per segment varied from 5 to 16, and the percentage of regeneration between 70 and 100%, depending on the genotype tested and the maturity of the explant. Regeneration capacity increased dramatically from the tip towards the base of the leaf, and was higher from the middle to the proximal end.Cefotaxime and carbenicillin, two antibiotics commonly used during transformation studies to eliminate Agrobacterium tumefaciens from plant tissue, were tested to determine their effect on morphogenesis. Cefotaxime at a dose of 250 mg 1^-1 enhanced regeneration and shoot development, whereas carbenicillin at a dose of 500 mg l^-1 induced abundant callus formation and inhibited regeneration. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. Schemes for selection and recovery of transgenic apple plants when kanamycin is used as the selection agent are discussed.Abbreviations BA benzyladenine - Cef cefotaxime - Crb carbenicillin - IBA indolebutyric acid - Kan kanamycin - LS Linsmaier and Skoog (1965) medium - M Malling - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - N6 medium (Chu et al. 1975) as modified by Welander (1988) - PPF photosynthetic photon flux     

In vitro axillary shoot proliferation of apple rootstocks under different ethylene conditions

Summary Ethylene effect on in vitro shoot proliferation of two apple rootstocks, MM111 and M9, was studied. Ethylene biosynthesis was proportionally stimulated by increasing concentrations of the precursor 1-aminocyclopropane-1-carboxylic acid (ACC). When 25 μM or more ACC was applied without any control of the headspace of culture vessels, shoot proliferation of both rootstocks was negatively affected. However, when shoot cultures were transferred to ACC-supplemented medium after the second week of culture, ACC had no effect. Supplementing the medium with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, together with the application of gas traps inside the flasks, significantly enhanced axillary shoot formation and elongation. Steady and high exogenous concentrations of ethylene in the culture flasks had negative effects on shoot proliferation. MM111 appeared to be more sensitive to ethylene than M9. For AVG a threshold dose was noticed, beyond which phytotoxic effects were induced.     

Stomata of apple leaves cultured in vitro

Examination by scanning electron microscopy showed abaxial stomata on in vitro cultured apple (Malus pumila Mill.) leaves. With leaf ontogeny, most of these stomata appeared to lose their regulatory ability while developing wide vestibules of up to 20 m in diameter. It is proposed that these deformed stomata may be a possible cause for the excessive transpirational water loss and consequent dehydration associated with transferring plants regenerated in vitro from culture.     

Dissecting apple tree architecture into genetic, ontogenetic and environmental effects: QTL mapping

The present study aimed to dissect tree architectural plasticity into genetic, ontogenetic and environmental effects over the first 4 years of growth of an apple F1 progeny by means of quantitative traits loci (QTL) mapping. Both growth and branching processes were phenotyped on the consecutive annual shoots of different axes within a tree. For each studied trait, predicted values (best linear unbiased predictors, BLUPs) of the genotypic (G) effect or its interaction with tree age (G×A) and climatic year (G×Y) were extracted from mixed linear models of repeated data. These BLUPs, which are independent from autocorrelations between repeated measurements, were used for QTL mapping. QTL detection power was improved by this two-step approach. For each architectural process, numerous QTLs were detected and some particularly interesting co-localised in common genomic regions, for internode lengthening, top diameter, and number and percentage of axillary shoots. When several QTLs were detected for a given trait, global models were estimated, which explained a maximum of 40% of the total variance for both internode length and top diameter and 28% for branching. QTLs detected for BLUPs of G×Y effects were interpreted as resulting from the interaction between genetic maximal potential of growth and climatic factors, while those for G×A effects were interpreted in relation to tree ontogeny. Most of the latter ones were found to be concomitant with key development stages during which the trait average started to decrease, but with different magnitudes depending on genotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Repeat flowering in apple caused by water stress or defoliation

Summary A series of experiments involving defoliation or water stress at different dates indicated that either of these treatments can make potted apple trees flower a second time in any one year, as long as the treatment is given near the end of July. The results suggest that the reflowering after a period of water stress was primarily a result of the loss of leaves that occurred when the plants were subsequently rewatered. Reflowering normally occurred only if flower primordia had already differentiated at the time of the treatment. There was an indication that in early July water stress was more effective than defoliation at stimulating reflowering.     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in photosynthetic characteristics during leaf development in apple

A comprehensive developmental survey of leaf area, chlorophyll, photosynthetic rate, leaf resistance, transpiration ratio, CO₂ compensation point and photorespiration was conducted in apple. The largest changes in each of the photosynthetic characteristics studied took place during the earliest stages of leaf development, coinciding with the period of greatest leaf expansion and chlorophyll synthesis. During early development, photosynthesis increased 5-fold, reaching a maximum rate of 40 mg CO₂ dm^-2 hr^-1 at a leaf plastochron index (LPI) of 10. During this same period, leaf resistance, transpiration ratio, CO₂ compensation point and mesophyll resistance decreased, while carboxylation efficiency increased. Two especially interesting aspects of the data discussed are simultaneous changes that occur at a LPI of 10 and 12 in all of the photosynthetic characteristics examined and an apparent decrease in photorespiration as leaves age. From our results it is clear that stage of leaf development is an important factor affecting the rate of photosynthesis and photorespiration.Scientific Paper No. 5687, College of Agriculture, Washington State University, Pullman. This work is supported by the National Science Foundation Grant 80-10958 and the Columbia River Orchards Foundation.     

Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg. JMS and SGJ contributed equally to this work     

Toxicity of ethanol during proliferation and adventitious root formation in apple microcuttings in vitro

We have examined the toxicity of ethanol in tissue culture of the apple rootstock ‘Jork 9’. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal correlations of specific leaf weight to net photosynthesis and dark respiration of apple leaves

Specific leaf weight (SLW), net photosynthesis (P _n ), and dark respiration (R _d ) of apple leaves were monitored for an entire growing season. Leaves were sampled from the canopy interior and periphery to provide a range of SLW. Leaf P _n was linearly correlated with SLW until mid-August, when P _n began to decline. During September the relationship between SLW and P _n was a quadratic. Leaf R _d and SLW were linearly correlated throughout the season. Leaf P _n and R _d were significantly correlated through most of the season, but the relationship was not always linear. Specific leaf weight appears to be a reliable index of the previous light environment of a leaf, but use to estimate P _n is probably limited to the first half of the season, because of increased variation after mid-August.Former Graduate Research Assistant (presently Assistant Professor, Department of Horticulture and Forestry, Rutgers University, Cook College, New Brunswick, NJ 08903, USA) and Associate Professor, respectively.