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     

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.     

Strong synergistic effects of gibberellins with the synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea on parthenocarpic fruit set and some other fruit characteristics of apple

The induction of parthenocarpic fruit set was investigated using the apple cvs. Golden Delicious and Jonagold. The gibberellins GA₃, GA₄, GA₅ and GA₇ and the synthetic phenylurea-type cytokinin CPPU (N-(2-chloro-4-pyridyl)-N-phenylurea), were applied alone and in combination to unpollinated flowers at the end of petal fall. Gibberellins induced only a marginal final set of parthenocarpic fruits. CPPU sprays were more effective, particularly in the first year. When applied in combination, CPPU and gibberellins had a positive synergistic effect on parthenocarpic fruit set and fruit size, but a negative effect on flower induction the next year. After CPPU + GA sprays, percent fruit set was similar, or greater, compared to natural pollinated trees. The parthenocarpic fruits induced by CPPU + GA had an increased length to diameter ratio. CPPU stimulated, and GA₄ and GA₇ reduced, the russeting of the parthenocarpic fruits. The internal quality of the fruits was hardly affected, but Ca-deficiency symptoms occurred more frequently in parthenocarpic fruits.     

Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa)

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstar and Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi (Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.Abbreviations BAP 6-Benzylaminopurine - E Elstar - H Holsteiner Cox - HPLC High performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron - YEP Yeast extract broth Communicated by H. Lörz     

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.     

苹果果实糖积累特性与品质形成的关系

以'富士'和'国光'苹果为研究对象,对其果实发育过程中糖含量及其代谢关键酶活性的变化进行测定分析,以揭示糖分积累代谢特性对果实品质形成的影响.结果表明:(1)'富士'和'国光'均为己糖积累型果实, '富士'果实以积累果糖最多,果糖/葡萄糖(F/G)值为1.56,而'国光'以积累葡萄糖最多,F/G值仅为0.68;蔗糖在两品种中含量和所占比例均很低,在近成熟期'富士'高于'国光'.(2)'富士'果实蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)活性均随果实糖的累积量增加而显著升高,酸性转化酶(AI)活性也渐趋升高,而中性转化酶(NI)活性波动不大,且其糖累积与AI和SPS活性相关性最大,而与NI相关性不大,SS的作用主要表现在发育后期;在 '国光'果实糖积累过程中SPS起主导作用,SS和NI的作用主要表现在发育前期,而AI的作用不大.(3)'富士'和'国光'果实淀粉含量变化趋势相同,在淀粉积累高峰之后,'富士'果实淀粉降解速度更快,其淀粉含量迅速下降且低于'国光',此时其相应淀粉酶活性也高于'国光'.研究发现,'富士'和'国光'果实糖积累和淀粉代谢均存在显著差异,从而直接或间接地影响着果实糖代谢过程,进而导致果实品质的显著差异.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

In vitro test of self-incompatibility in Malus domestica

Summary An in vitro assay in which self-incompatible pollen of Malus domestica is selectively inhibited is described. This assay involves heat-labile substances diffusing from the stylar tissues — in particular, glycoproteins found in the protein extract of styles. In the presence of the self-style extract, a dramatic decrease in total protein concentration in the culture medium was revealed at 30-min germination. Pretreatment of the self-pollen with 100 mM glucose prevented this drop in protein level; moreover, tube growth was entirely restored. A possible explanation in terms of protein-carbohydrate complementation is suggested.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

5-氨基乙酰丙酸对苹果叶片耐弱光能力的影响

为了探讨5-氨基乙酰丙酸(ALA)对苹果耐弱光性的影响,以‘润太2号’和‘郑优3号’两个品种苹果为材料,设置露天对照(CK)、轻度遮荫(LS)和重度遮荫(SS)3种光照条件,通过根际浇灌法研究了10mg·L~(-1) ALA处理对弱光条件下苹果叶片活性氧代谢与叶绿素荧光特性的影响。结果显示:(1)与CK相比,弱光胁迫显著降低了两品种苹果叶片的超氧化物歧化酶(SOD)与过氧化物酶(POD)的活性,增大了超氧阴离子(O_2~(-·))产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量,且‘郑优3号’的活性氧产生速率及MDA含量在弱光下的升高幅度更大;ALA处理显著提高了弱光胁迫下两品种的保护酶活性,降低了活性氧产生速率和MDA含量,并以耐弱光性较差的‘郑优3号’的变化更显著。(2)在弱光胁迫下,苹果叶片的叶绿素含量和叶绿素b/a升高,而ALA处理使二者进一步显著升高。(3)弱光胁迫下,苹果叶片的叶绿素荧光参数V_J、M_o、DI_o/RC显著升高,而ψ_o、ψE_o、ψR_o、PI_(ABS)、PI_(CS)和PI_(total)显著降低;ALA处理抑制了叶绿素荧光参数在弱光胁迫下的变化,甚至使其达到优于对照的水平。研究表明,弱光条件下苹果的抗氧化能力较差,受到明显的氧化伤害,且‘郑优3号’的耐弱光能力比‘润太2号’差;ALA处理提高了苹果在弱光下的抗氧化能力,降低了弱光对苹果叶片的氧化伤害,同时还提高了弱光条件下苹果叶片捕捉、传递和转化光能的效率,改善了光合电子传递情况,增大了苹果叶片在弱光胁迫下的光能利用效率,改善了光合性能;根灌ALA可以明显提高苹果的耐弱光性。     

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a β-glucuronidase (GUS) gene was transferred into apple cv. ‘Holsteiner Cox’ via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1–2 g l⁻¹ mannose in combination with 30 g l⁻¹ sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l⁻¹ and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.     

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.     

Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.     

Interaction of jasmonic acid with some plant growth regulators in the control of apple (Malus domestica) embryo germination

Embryos isolated from dormant apple seeds were treated with jasmonic acid (JA), gibberellin A₃ (GA₃), abscisic acid (ABA) and hydrogen cyanide in darkness and in light. The chemicals were present in the culture medium continuously and simultaneously or applied for 2 days and in different sequences. All treatments stimulated embryo germination except ABA, which was strongly inhibitory. Additive effects of JA with light and with GA₃ on embryo germination were observed, whereas ABA interacted synergically with JA, HCN and light. ABA and GA₃ were most effective when applied early during embryo incubation, but the late JA treatment was more stimulatory. It is concluded that JA does not act on the regulatory pathway that is initiated by light and which leads to embryo germination through gibberellin accumulation and alkaline lipase activation. ABA and HCN appear to be involved in the control of this pathway. JA and ABA may be involved in the control of alkaline lipase activity, independently of this regulatory chain.Abbreviations ABA abscisic acid - GA₃ gibberellin A₃ - JA jasmonic acid     

Self-fertile apple resulting from S-RNase gene silencing

Self-incompatibility (SI) restricts fertilisation and fruit setting in many tree fruit crops. In apple, we have produced transgenic trees harbouring extra copies of the endogenous S-gene controlling SI. Two independent transgenic genotypes were characterised in detail. Controlled self- and cross-pollination of the flowers of trees from both genotypes over a 3-year-period showed that the transgenic lines produced normal levels of fruit and seeds after selfing. In contrast, the controls produced much less fruit following self- compared to cross-pollination. Fruit set data correlated with the results of microscopic evaluation of pollen tube growth through the pistil, which revealed inhibition after selfing in the controls but not in the transgenic lines. The self-fertile phenotype was associated with the complete absence of pistil S-RNase proteins, which are the products of the targeted S-gene. These results confirm that self-fertility was due to inhibition of expression of the S-RNase gene in the pistil, resulting in un-arrested self-pollen tube growth, and fertilisation.Communicated by P. Debergh