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.     

Agrobacterium-mediated transformation of the apple rootstock Malus micromalus Makino with the RolC gene

Summary Malus micromalus Makino was genetically engineered with the rolC gene via Agrobacterium-mediated transformation. The antibiotics carbenicillin, hygromycin, and kanamycin (Km) inhibited shoot regeneration from in vitro leaf explants. The leaf segments were infected with Agrobacterium tumefaciens strain LBA4404 harboring one of the three different plasmids. Shoots were regenerated only from leaf segments infected with LBA4404 harboring the plasmid with the introncontaining neomycin phosphotransferase II gene as the selectable marker. Preculture of leaf segments on regeneration medium for 2 d before Agrobacterium treatment reduced formation of Km-resistant calluses. Transformation was confirmed by a histochemical β-glucuronidase (GUS) assay, amplification of the rolC gene by polymerase chain reaction (PCR), and by Southern blot analysis. The rolC-transformed M. micromalus shoots showed an increased rooting ability without auxin treatment, and reduced height, internode length and leaf areas. This research shows the potential application of using the rolC gene for developing dwarf apple rootstocks.     

Involvement of polyamines in the adventitious rooting of micropropagated shoots of the apple rootstock MM106

Apple rootstock MM106 shoots, raised in vitro, rooted at 96.7% after culture on a medium supplemented with an auxin for 5 d in darkness followed by culture on a second medium without growth regulators for 25 d in light. In control conditions (in absence of auxin in the first medium), these shoots did not root. Putrescine (PUT), spermidine (SPD), cyclohexylamine (CHA), and aminoguanidine (AG) enhanced rooting when applied during the first d of culture in the absence of IBA; on the contrary, α-difluoromethylornithine (DFMO) added to the first medium with IBA inhibited rooting. The endogenous levels of indole 3-acetic acid (IAA) and indole 3-acetylaspartic acid (IAAsp) increased up to a maximum concentration at days 2 and 3, respectively, in initial rooting conditions. PUT, when added with IBA, did not affect the typical IAA and IAAsp increase; when applied alone, it provoked an increase of their levels. Similar results were recorded with CHA. SPD, AG, and DFMO did not induce an increase of IAA and IAAsp in nonrooting conditions. The levels of endogenous PUT increased to a maximum at day 2 in rooting conditions; it was slightly affected by exogenous PUT and CHA application but reduced by SPD, AG, and DFMO. In rooting conditions, if the first medium was supplemented with SPD or AG, a small increase in peroxidase activity was observed, similar to that obtained with PUT treatment. The present work indicates an involvement of polyamines in the control of rooting and an interaction with auxins during the physiological phase of rooting. The consequence of this relationship was a different rooting expression, according especially to the content of these regulators in the culture medium.     

Phytophthora collar rot of apple: influence of the rootstock on scion variety resistance

In field experiments with young trees great differences were found in the resistance to Phytophthora cactorum of Cox's Orange Pippin apple scions grafted on different clonal rootstocks. The rootstock effect on scion resistance was inversely related to the effect on tree vigour: the rootstocks inducing high resistance were dwarfing (M. 9) or semi-dwarfing (M. 7, M. 26, MM. 106), and those inducing low resistance were vigorous or very vigorous (M. 25, MM. 104, MM. 109). Mean lesion lengths in Cox on MM. 104 were five to eight times greater than those in Cox on M. 9. The rootstock influence on scion resistance was associated primarily with effects on the rate of lesion extension: during the early stages only of host colonization there appeared to be threshold extension rates below which host resistance factors effectively suppressed a large proportion of infections. The influence of the root-stock on scion resistance was apparently unrelated to inherent rootstock resistance. On all rootstocks Cox showed diminished resistance to infection during the period from the swelling of buds to the early stages of shoot growth. Although most susceptible during the ‘mouse-ear’ and ‘pink bud’ stages of development, suscpetibility was not associated with flowering per se. Rootstock type did not affect the resistance of Cox scions to P. syringae, for which the period of susceptibility to infection occurred in the dormant season.     

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.     

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     

Successful propagation in vitro of apple rootstock MM106 and influence of phloroglucinol.

Successful in vitro propagation of clonal apple rootstock MM106 was achieved by culturing axillary buds on MS basal medium with BAP (1 mg/L), GA3 (0.5 mg/L) and IBA (0.1 mg/L). Use of liquid medium (LM) in initial cultures reduced phenol exudation to a greater extent and gave maximum sprouting percentage when transferred to solid MS medium. Phloroglucinol (PG) did not enhance sprouting of buds but increased the rate of multiplication when added in the medium. Maximum number of shoots were obtained when MS medium was supplemented with BAP (0.5 mg/L), GA3 (1 mg/L), IBA (0.1 mg/L) and PG (100 mg/L). For rooting, in vitro regenerated shoots were placed in IBA (30 mg/L) for 3 hr and transferred to solidified auxin free medium. Rooting was recorded in about 80% of shoots. Inclusion of PG in rooting medium was not beneficial but shoot cultures grown in its presence gave higher rooting percentage. Rooted plantlets showed about 70% survival rate in potting mixture of sand:soil:perlite (1:1:1).     

The rooting ability of shoots raised 'in vitro' from the apple rootstock A2 in juvenile and in adult growth phase

The apple rootstock A2 can be readily propagated in vitro both in the juvenile and in the adult growth phase. Shoots were produced by meristem tip culture from the apple rootstock A2 in different growth phases. The influence of growth phases and different concentrations of PG and IBA was investigated as to rooting percentage, survival percentage, number of roots per rooted shoot, root length, shoot length and formation of callus. IBA at 15 μ M without PG gave a significantly lower rooting percentage than 5 and 10 μ M IBA. PG together with IBA stimulated rooting, the optimum concentrations of PG being, however, not the same for the different growth phases. For the adult growth phase, 10⁻⁴ M PG promoted rooting, whereas 10⁻³ M PG markedly inhibited rooting. In the juvenile growth phases, both 10⁻⁴ and 10⁻³ M PG stimulated rooting. PG at 10⁻⁴ M also increased the number of roots. The longest roots were obtained at 10⁻³ M PG and 5 μ M IBA. PG at 10⁻³ M reduced callus formation at all IBA concentrations used. Neither shoot length nor root length influenced the survival percentage.     

Possible roles of auxin and zeatin for initiating the dwarfing effect of M9 used as apple rootstock or interstock

Endogenous indole-3-acid (IAA) and zeatin (t-Z) may play important roles in the dwarfing mechanisms of rootstocks. The concentrations of IAA and t-Z, as well as the expression of the genes PIN1 and IPT3 were measured in leaves, barks, and roots from nine treatments: M9, Malus X micromalus Makino, Red Fuji/M9/Malus X micromalus, Red Fuji/M9, Red Fuji/Malus X micromalus, M9 rootstock substitution above and below the original graft union, interstock bridging, and interstock bark substitution of M9. The results show that there were greater amounts of t-Z and expressions of IPT3 in invigorated trees (Malus X micromalus and Red Fuji/Malus X micromalus) when compared with dwarfing trees (M9, Red Fuji/M9/Malus X micromalus and Red Fuji/M9) during the period of fast shoot growth (early June to mid-August). Moreover, the variation of IAA contents and PIN1 expressions shared the same pattern with t-Z contents and IPT3 expressions in all the tested locations. IAA content was extremely high in the bark of M9 interstock when compared with that of the scion and the rootstock, yet PIN1 expression in corresponding tissues was very low. After M9 rootstock were substituted above and below the original graft union, contents of t-Z, IAA, and PIN1 expressions in leaves and branch-barks recovered gradually to standard tree levels. However, there is no significant difference between the two treatments. We conclude graft union has no obvious influence on hormone transport. After M9 interstock and its bark were substituted, the hormone measurement of every index was consistent with that of rootstock substitution.     

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.     

Effect of darkness treatment on the morphology,hormone status and gene expression of developing adventitious root in apple rootstock

Adventitious root (AR) formation plays a critical role for the clonal propagation of horticultural crops. In this study, Malus prunifolia var. ringo micro-cuttings were treated with IBA and darkness treatments to investigate the mechanism of darkness effects AR formation. Morphological and anatomical observation revealed that darkness promotes more ARs formation. Root morphology, and hormones level were evaluated during AR formation. Results indicated that darkness promotes AR formation by increasing auxin level at stages of 1 d and 3 d, moreover, ZR, ABA, GA₃, BR and JA also changed. The number of ARs in darkness-treated group was significantly higher than that of control treatment; Additionally, genes related to light signal pathway-, hormones-, carbohydrate metabolism-, cell cycle-, and root development were determined by RT-qPCR. Auxin- and cytokinin-related genes, such as MdARF1, MdGH3;1, MdPAT1-1; MdPIN1, MdCRF2, MdCRF4, and MdRR10 were significantly induced by darkness treatment, which corresponded with higher hormones levels. Furthermore, light-signal-related genes (MdASP3, MdATPC1, MdBGLU13, MdCBA1, MdLHCB6, MdLHCA1, MdNPQ4, MdPTE1 and MdPSAL) were also induced by darkness-treatment, resulting changes in the expression profiles of cell cycle-related genes (MdCYCB2;1, MdCYCB2;3–1 and MdCYCB2;3–2) and also in the expression of root development-related genes (MdLRP1, MdRHS19, MdSGR1 and MdSHI). Collectively, darkness treatment mediated auxin-, cytokines-, light signal-, cell cycle-, carbohydrate metabolism-, and root development-related pathways to regulating AR formation. This work laid the foundation for applied research and could be useful in future crop improvement programs.

     

The effect of paclobutrazol on in vitro rooting and growth of GF-677 hybrid peach rootstock

This paper deals with the evaluation of the influence of paclobutrazol (N-dimethylaminosuccinamic acid) and 2-naphtoxyacetic acid on rooting and growth of GF-677 hybrid peach rootstocks. The influence of these substances on the average number of roots per plant, the average length of roots per plant and the height of plants was evaluated as well as the effect of the addition of paclobutrazol before and after media sterilisation. As the obtained results indicate, plants, which were rooted on media with paclobutrazol and without auxin had the lowest number of roots per plant. Paclobutrazol showed a statistically significant negative effect on both the length of roots and the height of plants. It canbe concluded that, for the rooting of GF-677 rootstock it is helpful to use auxin plus paclobutrazol in concentration 0.43 μM. Higher concentrations affect inhibition, mainly in height of plants 14 days after transplant to soil. A part of the results referring to the influence of PP 333 on numbers of roots was presented at the conference “Recent Advances in Plant Biotechnology, 5th International symposium in the series”, Stará Lesná, Slovakia, 7–13 September, 2003.     

The effect of Fe-EDDHA and of ascorbic acid on in vitro rooting of the peach rootstock GF-677 explants

The objective of this research was to study the effect of the chelated form of the iron salt of ethylenediamine di-o-hydroxyphenylacetic acid (Fe-EDDHA) (6% Fe) on in vitro rooting of the rootstock GF-677. The iron salt of ethylenediamine tetraacetic acid (Fe-EDTA) (12% Fe) of the MS basic medium was replaced by Fe-EDDHA, which was applied in three concentrations: 93.5, 187.0 and 280 mg l⁻¹ (5.6, 11.2 and 16.8 mg l⁻¹ Fe, respectively). For each treatment of Fe-EDDHA, the effect of ascorbic acid added in four concentrations (0, 0.1, 1.0 and 10 mg l⁻¹) was studied. After 4 weeks of culture, the explants growing on the medium with 280 mg l⁻¹ Fe-EDDHA gave the best rooting results. Regarding ascorbic acid, no clear stimulating effect on rooting was found.     

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.     

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.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The formation and subsequent growth of roots by cuttings of poinsettia, hydrangea, rose and azalea in various propagation media, Jiffy-7, Jiffy-9 and Grodan under different conditions of aeration was investigated. The interrelationships of the effects of air content of the media, temperature and light intensity on the rooting of poinsettia cuttings was also studied.With low air contents (0 cm moisture tension) in the propagation media the formation and growth of roots was strongly inhibited. The rooting performance of rose appeared to be less affected by the poor aeration. Increasing air content improved rooting but best results were obtained at moisture tensions of 4 to 8 cm. Rooting seems to be better correlated with oxygen diffusion rate (ODR) than with air content.For poinsettia cuttings the optimum temperature for rooting was 24 to 28°C. At low temperatures rooting was delayed while at higher temperatures it was almost completely inhibited. Callus formation increased with temperature but decreased with increasing moisture tension. Conditions which induced large callus formation inhibited root formation.High light intensity during rooting reduced overall rooting performance and the inhibition was most pronounced in conjunction with high moisture tensions.Report No. 255.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The air content in three types of propagation media, Jiffy-7 and Jiffy-9 which are Sphagnum peat and Grodan which is rockwool, were investigated when they were held at moisture tensions of 0,6 and 12 cm measured from the base of the media. At 0 cm tension the air content (vol. %) was highest in Jiffy-9 and lowest in Jiffy-7. At 12 cm tension the air content was higher in Grodan than in Jiffy-9 and Jiffy-7. Oxygen diffusion coefficients (ODC) and oxygen diffusion rates (ODR) were measured at the different air contents. At air contents below 20 vol. % ODC was about the same for Jiffy-9 and Grodan but at air contents above 20 vol.% it was larger for Jiffy-9 than for Grodan. The oxygen diffusion rate was measured at 0, 4 and 8 cm moisture tension. At all tensions it was approximately 20% higher in Jiffy-9 than in Grodan and Jiffy-7. The ODR in Jiffy-7 and Grodan were affected equally at the same tension, although Grodan contained more air. Report no 253     

渭北旱塬矮砧密植苹果园土壤矿质氮积累与空间分布特征

随着苹果矮砧密植栽培模式的迅速发展,揭示矮砧密植苹果园土壤矿质氮的积累与分布特征对果园科学施肥具有重要意义.本研究以不同树龄(6a、9a、12a)的矮砧密植苹果园为对象,在树下、株间、行间以及树干与行间的中间点位置采集土样,分析土壤硝态氮、铵态氮和矿质氮的积累与分布特征.结果 表明:O～ 300 cm土层土壤硝态氮累积...