Brassinosteroid-stimulated branch elongation in the marubakaido apple rootstock

Brassinosteroids (BRs) comprise a specific class of low-abundance plant steroids now recognized as a new class of phytohormones. In this paper, we demonstrate that a fluoro derivative of 28-homocastasterone (5F-HCTS) stimulates branch elongation in in vitro-grown shoots of Malus prunifolia, the marubakaido apple rootstock. In addition to that, we show that this BR-stimulated branch elongation is paralleled by an increase in ethylene release. However, either the presence of 1-amino-cyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in higher plants, in the culture medium or an ethylene-enriched atmosphere resulted in inhibition of branch elongation, indicating that the stimulation of branch elongation observed for 5F-HCTS-treated shoots in this study was not, at least directly, related to the BR-induced enhancement in ethylene release rate. Besides its positive effect on the marubakaido shoot growth, i.e. branch elongation, the 5F-HCTS-driven enhancement of branch elongation found in this study is potentially useful to improve micropropagation techniques for other plant species as well, especially woody species, in which branch elongation is typically a constraint for efficient micropropagation.     

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

Differential performance of marubakaido apple rootstock shoots grown in culture media containing different agar brands: dynamic rheological analysis

Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents. In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921 agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921 and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance, i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars.     

Influence of arginine onin vitro rooting of dwarf apple rootstock

L-arginine was added to the rooting media for apple rootstock shoots taken from proliferating cultures. The effect was studied in combination with other rooting factors such as: phloroglucinol, initial dark period, concentration of indol-3-yl butyric acid and inorganic nitrogen levels. In all treatments, arginine caused an increase in root number per rooted shoot and enlargement of the shoot base. Arginine was especially effective with low indol-3-yl butyric acid levels as well as without it, and with low or no inorganic nitrogen. The effect of arginine on root number interacted with dark treatment and with phloroglucinol. The most efficient amount of arginine was 200 mg l^–1. The possible influences of arginine on rooting are discussed.     

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).     

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.     

Enhancement of in vitro shoot regeneration from leaf explants of apple rootstock G.41

Apple (Malus domestica) rootstock G.41 is an excellent member of the Geneva series because it has traits for resistance to abiotic and biotic stresses. A simple and efficient protocol for obtaining shoots from leaf explants was established by optimizing the combinations of plant growth regulators, mode of wounding, and explant orientation on the culture medium. The best shoot multiplication index (2.58) was obtained from successful subculture medium that was the standard Murashige and Skoog (MS) medium supplemented with 7.5 g L^?1 agar, 3.55 μM N ⁶-benzyladenine, 0.16 μM indole-3-butyric acid, and 30 g L^?1 sucrose. Regeneration rates were highest (99%) when MS medium was supplemented with 2.7 μM thidiazuron and 0.9 μM 1-naphthaleneacetic acid, and cut-wounding explants before placing the abaxial surface in contact with the medium. The best rooting percentage (80%) was obtained on MS medium supplemented with 4.92 μM indole-3-butyric acid. Plantlets were rooted in vitro and survived acclimatization in the laboratory and greenhouse.     

An analysis of mineral uptake in apple rootstock seedlings

Summary Eight families from biparental crosses of apple rootstocks and 12 families from open pollinated Malus spp. were analysed in 2 years for N, P, K, Ca and Mg content of the foliage. Highly significant differences were found between the families for all elements. There were no significant differences between the means of the biparental group and the open pollinated group. Ca and K content were significantly more variable in the open pollinated families compared with the biparental families. It is suggested that this increased variation could prove useful in breeding for efficiency of mineral uptake by apple rootstocks.     

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.     

Proliferation enhancement by spontaneous multiplication of chromosome 7 in rheumatic synovial cells in vitro

Mosaic trisomy of chromosome 7 is known to occur in a variety of non-neoplastic hyperproliferative disorders. In long-term cell cultures established from rheumatic synovium with mosaic trisomy 7, we observed a continuous increase in the proportion of cells with trisomy 7 to over 50% by the 10th in vitro passage. Simultaneous in situ hybridization with a repetitive chromosome-7-specific DNA probe and fluorescent Ki-67 labelling showed a strong correlation between trisomy 7 and an elevated proliferation index in cultured rheumatic synovial cells. Moreover, we observed a fraction of rapidly proliferating cells with up to eight copies of chromosome 7 as the sole cytogenetic change. Frequent somatic pairing of centromeres of two chromosomes 7 in interphase nuclei suggests either atypical non-disjunction with a persisting centromere or selective endoreduplication of chromosome 7.     

Excised rootstock roots cultured in vitro

Root cultures have been established successfully for the Prunus rootstocks Adafuel and Adarcias (Prunus×amygdalopersica), A843 (P. armeniaca), Mariana 2624 (P. cerasifera×munsoniana) and Myrobalan 605AD (P. cerasifera), and for the apple rootstock Jork 9 (Malus×domestica). High percentages of root tips grew during the first 15 days and then decreased. Root growth was affected by culture conditions and the composition of the culture medium. Liquid medium was preferable as increasing agar concentrations reduced root growth. Darkness, instead of a 16-h photoperiod, was beneficial for Adafuel, but not the other genotypes. Sucrose at 3% was better than higher concentrations. Full Murashige and Skoog salts sustained better root growth than dilutions to 1/2 or 1/4. The addition of various organic supplements such as coconut water, casein hydrolysate or malt extract did not improve root growth, and sucrose was the best carbon source tested. Data presented here support the notion that excised root culture is an efficient experimental model to study the response to various factors, since controlled variations in the culture medium, such as those studied here, had a very noticeable effect on root length. Received: 4 June 1997 / Revision received: 3 February 1998 / Accepted: 1 March 1998     

Characterization of bacteria contaminating tissue cultures of apple rootstock 'YP'

Meristem-tip cultures of apple rootstock 'YP' were started at different times of the year over a period of 2 years and the contamination of the cultures was monitored during five subcultures. Bacterial contaminants were isolated to pure cultures, identified by the API test system and appropriate additional tests, and the sensitivity of the most common isolates to different antibiotics was determined. Of the 216 strains isolated from the initiation cultures, 78% were pseudomonads, coryneforms or enterobacteria. Only three bacterial contaminants were found at the multiplication stage. A greater part of the contaminants were likely to originate from the stock plant. Rifampicin (at 50–200 mg 1^-1) and cefotaxime (at 250–1500 mg 1^-1) were found to be bactericidal against many isolates, but differences between species and strains were found.     

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.     

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

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.     

Plantlets from encapsulated micropropagated buds of M.26 apple rootstock

In order to be considered usable as synthetic seeds, encapsulated explants sown underin vitro orex vitro conditions must be able to produce whole plantlets. Ninety percent of non-encapsulated M.26 apple rootstock single nodes produced a plantlet (i.e., a well-formed shoot with a root system) after 30 days of culturein vitro if the explants were previously given a 24-hour treatment with 24.6 μM IBA and 15 g 1⁻¹ sucrose in darkness. In contrast, when the single nodes were encapsulated in a calcium-sodium alginate bead immediately after the same treatment only 10% of the encapsulated explants formed a plantlet. Addition of growth regulators to the artificial endosperm and culture of the single nodes for root primordia initiation for 3, 6 or 9 days in darkness before encapsulation allowed production of 58%, 60% and 66% of plantlets, respectively.     

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     

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.