Application of architectural analysis and AMAPmod methodology to study dwarfing phenomenon: the branch structure of 'Royal Gala' apple grafted on dwarfing and non-dwarfing rootstock/interstock combinations

Architectural analysis was applied to study branch development of 'Royal Gala' apple trees grafted with dwarfing and non-dwarfing rootstock/interstock combinations, which had been chosen to produce trees with a wide range of vigour. Using AMAPmod methodology, the structure of 3-year-old branches was described at four levels of representation: branch; annual shoot; growth unit; and node. Three types of growth units were distinguished: extension growth unit (vegetative unit with internode extension); vegetative spur with minimal internode extension; and fruiting spur or bourse. The aim of the analysis was to describe exactly how the rootstock/interstock combinations affected the structure building process. The number of extension growth units, vegetative spurs and fruiting spurs per annual shoot changed over the years, but this was not affected by rootstock/interstock combination. Compared with MM.106 rootstock, M.9 rootstock reduced the number of nodes per extension growth unit. In most cases, rootstock/interstock combination had no effect on the linear relationship between extension growth unit length and node number (R(2) = 0.88). Average internode length depended on unit node number, with internodes being shorter for units with fewer nodes. Thus the difference in apple branch size induced by the rootstock/interstock combinations was mainly due to a reduction in the length and number of neoformed nodes produced on extension growth units. As percentage budbreak of axillary buds on extension growth units was not affected by rootstock/interstock combination, differences in numbers of axillary annual shoots per branch were entirely due to differences in the total numbers of nodes extended during the previous year.     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

Propagation in vitro of the apple rootstock M4: effect of phytohormones on shoot quality

The quality of shoots in cultures of the apple rootstock, M4, was used as a criterion for the selection of an optimum medium. The frequency of shoots in defined shoot clases was monitored for each of five media, which differed in the type and concentration of phytohormone. Media containing BA (1.15 mg l^-1) and IBA (either 0.15 or 0.20 mg l^-1) produced the maximum number of shoots that were desirable for transplantation and acclimatization.     

Diheteroarylurea derivatives as adventitious rooting adjuvants in mung bean shoots and M26 apple rootstock

The present research investigates the biological profile of eight symmetrical diheteroarylureas and phenylheteroarylureas, testing their hypothetical cytokinin-like activity and rooting activity. Cytokinin-like activity was assayed by the betacyanin (so-called amaranthin) accumulation test and by the tomato regeneration test. The rooting activity was assessed using the mung bean rooting test, the apple stem slice test and the rooting of apple microcuttings. Three compounds, 1,3-di(pyrazin-2-yl)urea (3a), 1,3-di(benzo[d]oxazol-5-yl)urea (3b) and 1,3-di(benzo[d]oxazol-6-yl)urea (3c), enhanced adventitious root formation in apple stem slice test, but only 3b and 3c were active in the mung bean rooting test. Compound 3b, that showed the best rooting activity, was also able to enhance the adventitious root formation in apple microcuttings. None of the compounds showed cytokinin-like activity.     

不同树盘覆盖对矮砧苹果园土壤微生物群落结构和多样性的影响

以7年生矮化中间砧苹果为试材,研究了不同材质树盘覆盖对土壤微生物丰度、群落结构和多样性的影响。高通量测序结果表明,防水园艺地布和塑膜覆盖处理提高了0-20 cm土层变形菌门的相对丰度和20-40 cm土层变形菌门的相对丰度,防水园艺地布处理还显著提高了土壤细菌Ace和Simpson指数。相较于防水、塑膜覆盖处理,透水园艺地布不仅提高了0-20 cm土层变形菌门、接合菌门的相对丰度,而且提高了土壤细菌Ace、Chao1和Simpson指数,降低了土壤真菌Ace、Chao1和Simpson指数。树状聚类图结果显示,树盘覆盖显著改变了土壤细菌和真菌群落结构,以透水园艺地布覆盖处理的改变最为显著。综合分析认为,不同覆盖措施均有利于提高土壤细菌群落的丰富度和多样性,降低土壤真菌群落的丰富度和多样性,优化土壤微生物群落结构,其中以透水园艺地布覆盖的效果最好。     

Clarifying the effects of dwarfing rootstock on vegetative and reproductive growth during tree development: a study on apple trees

BACKGROUND AND AIMS: Despite the widespread use of dwarfing rootstocks in the fruit-tree industry, their impact on tree architectural development and possible role in the within-tree balance between growth and flowering are still poorly understood, in particular during the early years of growth. The present study addressed this question in apple trees, through a detailed analysis of shoot populations, i.e. both vegetative and flowering shoots, during tree development. METHODS: Architectural databases were constructed for trees of two cultivars that were either own-rooted or grafted on dwarfing rootstock. Within-tree shoot demographics and annual shoot characteristics, i.e. their dimensions, number of laterals and flowering, were observed from the first to the fifth year of growth and compared among scion/root system combinations. KEY RESULTS: Differences in axis demographics appeared among scion/root system combinations after the second year of growth. Differences were found (a) in the number of long axes and (b) the number of medium axes. Dwarfing rootstock reduced the total number of axes developed in a tree, and this reduction resulted from proportionally more medium axes and spurs than long axes. The life span of spurs was also shortened. These phenomena appeared after an increase in flowering that started in the second year of growth and involved both axillary and terminal positions. Flowering regularity was also increased in grafted trees. CONCLUSIONS: These results confirm that the number of long shoots and flowering potential depend on the cultivar. They indicate that tree architectural plasticity in response to its root system mainly derives from the number of medium shoots developed and follows priorities within the whole tree axis population. There was also evidence for dwarfing rootstock involvement in adjusting the flowering abundance and that differences in flowering occurrence take precedence over those regarding vegetative growth during tree development.     

The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26

 The apple rootstock M26 (Malus domestica) was infected with a binary vector system of Agrobacterium tumefaciens carrying the neomycin phosphotransferase II and Arabidopsis phyB genes. Thirteen transformed clones were obtained from 329 infected leaves. Five of the clones had a single copy integration, six clones had two copies, one clone had five copies and one of the clones had eight copies of the phyB gene integrated. No differences in rooting were found between transformed and untransformed plants. The stem length was reduced in nine of the 13 transgenic clones, and shoot, root and plant dry weights were reduced in all transformed clones compared with untransformed control plants. Northern analysis showed that the Arabidopsis phyB gene was expressed in the transformed clones. Received: 28 April 1999 / Revision received: 28 February 2000 / Accepted:29 February 2000     

Uptake and translocation of paclobutrazol by shoots of M.26 apple rootstock

When ¹⁴C-paclobutrazol, a gibberellin synthesis inhibitor, was applied to different parts of actively-growing M.26 apple rootstock shoots it was translocated acropetally when applied to the young stem and, to a lesser extent, from the youngest unrolled leaf. Paclobutrazol was not translocated out of leaf laminae, shoot tips or from one-year-old wood but translocation occurred out of a treated petiole into the attached leaf. No basipetal translocation was detected. This translocation pattern suggested movement through the xylem.Localised application of paclobutrazol caused a reduction in shoot extension and leaf production when the young stem or shoot tip were treated; the effect decreased as older parts of the stem were treated. Treatment of laminae or petioles had only a slight effect on shoot extension and treatment of one-year-old wood was ineffective. Combined treatment of the shoot tip plus young stem was similar in effect to treatment of the complete shoot.It is suggested that paclobutrazol exerts its effects on shoot growth by inhibiting gibberellin biosynthesis in the shoot tip and the expanding leaves.The findings contribute to an understanding of the requirements for efficient orchard application of foliar sprays of paclobutrazol.     

Physiological dissection of blue and red light regulation of apical dominance and branching in M9 apple rootstock growing in vitro

This paper presents the results of the interaction of red light (R) with blue light (B), applied to shoots of M9 apple (Malus pumila paradisiaca Schmid) rootstock, on the regulation of stem growth, apical dominance and branching. These results are compared with the active form of phytochromes (PHYs) under monochromatic and dichromatic light treatments. The inhibition of internode elongation was determined by PHY photoequilibrium, with the additional effect of B, by means of a separate photoreceptor. The development of phytomers appeared to be primarily due to the active form of PHY, with a marginal effect from B. Shoot growth, which combines internode elongation and development of the phytomer, was highest under R and lowest under B and far red light (FR), showing the largely positive role of PHY photoequilibrium. Under FR, reduced stem elongation was due to the very small number of phytomers formed. Apical dominance was inhibited, while branching was increased under R, corresponding to the highest values of PHY photoequilibrium determined. Apical dominance was increased and branching was reduced by B, indicating a complex interaction between PHY and B receptor. In the shoot cluster system, photomorphogenic behavior was dependent on the time of exposure to the different light qualities. The information gained from the study will be helpful in improving the set up of in vitro growth light conditions, and in providing useful insights into research of the development of the woody plant canopy, an important factor in ecological plant communities.     

Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting

A suitable protocol for transformation has been developed for the apple rootstock Jork 9 using Agrobacterium tumefaciens strain EHA101A(pEHA101A)(pSCV1.6). Root formation was increased by transforming the rootstock with A. tumefaciens strain C58C1(pGV3850)(pB-B:GUS), which contains the nptII, rolB and gus genes on the T-DNA. Transformation for all of the introduced genes was confirmed by polymerase chain reaction and Southern blot analyses. Of the 18 independent shoot lines obtained after transformation only ten contained at least one copy of intact T-DNA, while six lines were missing the gus gene and two lines were missing both the gus and rolB genes. The rooting experiments showed that introduction of the rolB gene increased root percentage and root number, giving 13.8 roots per shoot compared to 2.3 for untransformed shoots. More than two copies of the rolB gene decreased the number of roots and percentage of rooted shoots.     

Possible roles of calcium and ammonium in the development of bitter pit in apple

Apple trees ( Malus pumila Mill . var. domestica Fuji/ Malus prunifolia rootstock) showed a high susceptibility to bitter pit when supplyed with ammonium salt instead of nitrate (control) in the nutrient solution. When apple fruit was affected by bitter pit, a lower calcium as well as a higher nitrogen and ammonium-nitrogen contents was observed in the fruit flesh near the calyx end. The activity of the mitochondrial Ca²⁺-uptake of the fruit flesh near the calyx end was higher when the tree was grown with ammonium salt than when grown with nitrate. Both the activities of succinate: cytochrome c oxidoreductase and the mitochondrial Ca²⁺-uptake per g of tissue were higher in affected fruit than in healthy fruit. Each of chlorpromazine, N-(6-aminohexyl)-5-chloro-l-napthalenesulfonamide (W-7) and N-(6-aminohexyl)-l-naphthalenesulfonamide (W-5), calmodulin antagonists, was infiltrated into the fruit for 20 min under reduced pressure (about 1 × 10⁴ Pa). Few days later, numerous bitter pit-like spots were observed in both fruit treated with W-7 and chlorpromazine, while only a few spots were observed after the infiltration with W-5, a less potent calmodulin antagonist. A possible mechanism for the occurrence of bitter pit is discussed.     

Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle

The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4mol BAP and 0.5mol IBA during the shoot multiplication phase and 1.1mol BAP and 0.25mol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5cm or 1cm resulted in a significantly higher shoot production rate compared with that of 1.5cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse.     

High-frequency shoot regeneration from leaves of the apple rootstock M26 (Malus pumila Mill.)

Regeneration of shoots was achieved from in vitro leaves of M26 at frequencies close to 100% on a medium based on MS salts and LS vitamins, containing 4.4 M BA and 0.5 M NAA. Dark and red light gave the best results in inducing shoot regeneration. White light at high intensity helped development of regenerated shoots. Inorganic nitrogen could be reduced by 75% without negative effect, and the presence of NH₄ ⁺ was necessary for regeneration. Leaves were able to regenerate after a 3 kR irradiation (gamma rays), not after 4 kR. Optimal dose should be between 1 and 2 kR.Publication No 261, Centro Studi Tecnica Frutticola-CNR. Research work supported by CNR, Italy special grant IPRA. Sub-project 1, paper No. 1496.     

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

In vitro rooting of the apple rootstock M 26 in adult and juvenile growth phases and acclimatization of the plantlets

In order to obtain optimum conditions for in vitro propagation of the apple rootstock M 26 ( Malus pumila Mill.) in adult and juvenile growth phases, several rooting experiments were performed. Supraoptimal concentrations of indole-3-butyric acid (IBA) added to the rooting media resulted in profuse callus formation. Since extensive callus production is detrimental to the survival of the plantlets, modified culture conditions were established to reduce callus formation. A reduction of the time of exposure to IBA to 5 days and, thereafter, transfer to a hormone-free medium did not eliminate callus production. Exposure to darkness during the root initiation phase increased rooting. When the rooting medium was based on the Lepoivre formula instead of the Murashige and Skoog formula, callus formation was reduced. Optimum conditions for rooting were obtained at much lower concentration than earlier reported, being 1.25 μM for the juvenile and 0.5 μM for the adult growth phase in the range of IBA concentrations tested. Anatomical studies revealed that root initials are formed after 5 days of IBA-treatment. Therefore, we transferred shoots directly to non-sterile conditions after the root-inducing phase. This resulted in a 90% survival of the plantlets. Subculture on hormone-free medium can thus be eliminated when the optimum auxin concentration is known.     

Stigmasterol-driven enhancement of the in vitro multiplication rate for the marubakaido apple rootstock

The treatment of in vitro-grown shoots of the marubakaido apple rootstock with 0.5 μg stigmasterol, an end-pathway sterol of the bifurcated sterol biosynthetic pathway, in 5 μL acetone per shoot led to a significant (p ≤ 0.05) enhancement of the multiplication rate (MR) from 5.1 (shoots treated with 5 μL acetone only) to 10.3. This increase in the MR was due to a significant enhancement of the number of newly formed main shoots suitable for micropropagation purposes (measuring at least 15 mm in length) from 2.6 to 3.3 per explant, and of the number of newly formed primary lateral shoots from 2.2 to 5.0 per explant as well. Shoots treated with stigmasterol at 0.5 and 2.5 μg per shoot presented primary and secondary lateral shoots with significantly (p ≤ 0.05) longer length compared to shoots treated with acetone only. These results provide an insight into the morphological responses of the marubakaido rootstock shoots to the treatment with an end-pathway sterol. To the best of my knowledge, this is the first report on the successful use of stigmasterol for the improvement of a micropropagation system. These results also demonstrate that stigmasterol-induced shoot proliferation is a low-cost and effective way to enhance the in vitro MR for the apple rootstock.     

Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and commercial scion performance on Carrizo citrange rootstock

The term ‘transmissible small nuclear ribonucleic acids' (TsnRNAs) describes well characterised viroid RNA species that do not induce any disease syndromes in specific citrus hosts but rather act as regulatory genetic elements modifying tree performance. Twelve-year-old navel orange and 10-year-old Clementine mandarin trees on Carrizo citrange (Citrus sinensis×Poncirus trifoliata) rootstock treated with a mixture of three TsnRNAs (−Ia, syn. Citrus bent leaf viroid, +IIa, syn. Hop stunt viroid and +IIIb, syn. Citrus dwarfing viroid) were reduced in size by 33% and 43%, respectively. Clementine trees treated with a mixture of TsnRNA−Ia+IIa or −Ia+IIIb also had reduced canopy volume (CV) (∼38 and 31%, respectively), whereas trees treated with TsnRNA−IIa+IIIb showed little effect. The effects of the double TsnRNA treatments −Ia+IIa and −Ia+IIIb on Clementine canopy size and commercial performance were comparable and in some cases superior to that of the triple TsnRNA mixture. The TsnRNA−Ia+IIa treatment had the most attractive commercial traits with increased production of Clementine fruit per CV (23.6%), more fruit with high commercial value (31.7%), and more fruit optimally distributed in the canopy (68% of fruit between 0.5 and 2.5 m). None of the TsnRNA treatments affected the growth of Carrizo rootstock seedlings after 8 years in the field. Navel orange and Clementine scions treated with the same triple TsnRNA mixture expressed different trunk and fruit production patterns although effects on CV were similar.     

Micropropagation and synthetic seed in M.26 apple rootstock (II): A new protocol for production of encapsulated differentiating propagules

Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Several aspects of the technique are still underdeveloped and hinder its commercial application. One of these aspects is the high hand labor requirements and costly procedures for the production of encapsulated explants. Direct organogenesis is an efficient shoot regeneration method and uninodal explants can be used for encapsulated synseed production. In this paper, we compare the performance of encapsulated organogenetic explants of M.26 apple rootstock that were prepared by hand and mechanical manipulation. Conversion of synseed into plantlets was 25% from encapsulated hand-cut uninodal microcuttings and 11% from machine-ground explants. The results demonstrate that synthetic seeds of M.26 apple rootstock can be produced through organogenesis from machine processed explants followed by root induction and encapsulation of differentiating propagules.     

New roles for pyruvate kinase M2: working out the Warburg effect

The origins and role of the Warburg effect have remained uncertain for many years. Two recent studies demonstrate that an embryonic- and cancer-cell-specific isoform of the enzyme pyruvate kinase M2 (PKM2) is regulated by binding to phospho-tyrosine motifs and promotes increased cell growth and tumor development. PKM2 enhances the use of glycolytic intermediates for macromolecular biosynthesis and tumor growth. These findings illustrate the distinct advantages of this metabolic phenotype in cancer cell growth.     

Antioxidant and anatomical responses in shoot culture of the apple rootstock MM 106 treated with NaCl, KCl, mannitol or sorbitol

To determine whether the major influence of high salinity is caused by the osmotic component or by salinity-induced specific ion toxicity, we compared the effects of mannitol, sorbitol, NaCl and KCl (all in concentratuions corresponded to osmotic potential −1.0 MPa) on the antioxidant and anatomical responses of the apple rootstock MM 106 explants grown in the Murashige and Skoog (MS) medium. All the compounds had a significant influence on explant's mineral composition and reduced the leaf water content, whereas mannitol and salts decreased chlorophyll (Chl) content and increased proline content. Superoxide dismutase (SOD), peroxidase (POD) and non-enzymatic antioxidant activities as well as H₂O₂ content were increased in the leaves and stems. In addition, in the leaves of explants exposed to NaCl an additional Mn-SOD isoform was revealed, while specific POD isoforms were detected in the leaves and stems treated with NaCl or KCl. However, catalase activity was depressed in the salt-treated leaves. At the ultrastructural level, the NaCl-treated leaves had the thickest lamina, due to an extensive increase of the size of epidermal and mesophyll cells. Also, an increase of the relative volume of the intercellular spaces in response to NaCl was observed. The results suggest that Na accumulation is the first candidate for the distinct antioxidant and anatomical responses between saline and osmotically generated stress in the MM 106 explants.