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

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

Endogenous indole-3-acetic acid and abscisic acid in apple microcuttings in relation to adventitious root formation

The effects of applying indole-3-butyric acid (IBA) for periods up to 48 h were examined in difficult-to-root microcuttings (from newly-established cultures) and in easy-to-root microcuttings (from long-term subcultures) of Jonathan apple (Malus X domestica Borkh). In easy-to-root material, 20% of the microcuttings produced roots in the absence of IBA, while 6 h exposure to 10 M IBA gave 100% rooting of microcuttings. In contrast, root formation in difficult-to-root material was IBA-dependent. Maximum rooting of these microcuttings (50%) required 24 h exposure to 10 M IBA.Variation in the endogenous levels of free indole-3-acetic acid (IAA) during the course of root induction was similar in microcuttings of both types but there were marked differences in endogenous abscisic acid (ABA) levels. In easy-to-root microcuttings ABA remained at a constant low level, but in difficult-to-root material ABA exhibited marked fluctuations and was present at higher concentrations than in easy-to-root microcuttings.     

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT

We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9?s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.     

Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Anatomical and ultrastructural examination of adventitious root formation in stem slices of apple

Adventitious root formation in vitro in 1-mm stem slices cut from microshoots of apple cv. Jork 9 was studied using light and electron microscopy. When indole-3-butyric acid (IBA) had been added to the medium, starch grains accumulated during the first 24 h of culture in cells of the cambial region and in cells in the vicinity of vascular tissue and in the primary rays. This accumulation occurred only in the basal part of explants. After that, the nuclei in these cells were activated, and the density of the cytoplasm and the number of cell organelles increased, whereas starch was broken down. Cambium cells started to divide transversely and at 96 h, after several divisions, a continuous ring of isodiametric cytoplasmic cells had appeared around the xylem near the basal cutting surface. The cells in this ring were rich in cell structures, and did not contain large starch grains and a central vacuole. Root meristemoids regenerated from the portions of the ring that were localized in the primary rays. From the other cells in the ring, callus developed. The meristemoids did not grow into the direction of the epidermis as in shoots, but along the vascular bundles. After emergence from the cutting surface, the meristemoids were transformed into small, dome-like primordia. They developed a typical root apex with root cap, root ground meristem and tracheid connection with shoot vascular tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Timing the phases of the microtubule cycles involved in cytoplasmic and nuclear divisions in cells of undisturbed onion root meristems

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G₂ to prophase transition, ie it was only present during the final 5% of the total G₂ timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G₁ of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.     

Confirmation of the role of auxin and calcium in the late phases of adventitious root formation

Poplar shoots raised in vitro were induced to root by incubation on an auxin (NAA) containing medium for 7 h. After 13 days on an auxin-free medium, 97% of the treated shoots had rooted. The introduction of known antiauxins (PCIB, PBA, POAA) into the rooting expression auxin-free medium, after the 7-h induction by NAA, completely (PCIB and PBA) or severely (POAA) inhibited rooting. The exclusion of calcium from the expression auxin free medium reduced the percentage of rooting by about 42%. The inhibition was still higher in the presence of EGTA, a calcium chelator. Lanthanum chloride, a calcium channel blocker, also completely inhibited rooting, when incorporated into the auxin free medium, with or without calcium. These results support previous hypotheses about the implication of both endogenous auxin and calcium in the late phases of the adventitious rooting process.     

Effect of light and riboflavin on indolebutyric acid-induced root formation on apple in vitro

Maximum root formation on apple ( Malus ) shoots cultured in vitro occurred after an incubation in the dark on medium containing 3.2 or 10 μ M indolebutyric acid (IBA) plus riboflavin. Omission of riboflavin or culture in light resulted in a significant decrease in the number of roots formed. About 95% of the absorbed IBA was inactivated by conjugation, ca 4% was extracted as the free IBA acid (IBAH) and only 1% as IAAH. It was investigated whether the decrease in root formation caused by exposure to light or omission of riboflavin during culture was parallelled by a shift in the concentrations of the physiologically active auxin compounds (IBAH and IAAH) in the stem base, i.e. the location where the roots emerge. At least 90% of the absorbed ³H-IBA was located in the stem base. Omission of riboflavin, either in the dark or in the light, had no effect on the IBAH and IAAH concentrations, whereas root formation decreased significantly. Incubation in the light on medium containing 10 μ M IBA with or without riboflavin and culture in the dark on medium containing 3.2μ M IBA plus riboflavin resulted in similar IBAH and IAAH concentrations. However, the number of roots was significantly lower after culture in the light. Therefore, we conclude that the synergistic effect of riboflavin and the antagonistic effect of light on IBA-induced root formation are not solely based on changes in the concentrations of the active auxin components resulting from IBA uptake.     

Microphotography of phases in the development of apple flower buds

Je uveden vývoj květních pupen? jabloní od jejich vzniku na branchyblastech a? do doby krátce p?ed vykvetením. Celé období vývoje je mo?né rozdělit do ?ty? hlavních fází: I. fáze je vegetativní, v ní? vznikají na vzrostném vrcholu primordia krycích ?upin. II. fáze zahrnuje morfologiekou diferenciaci vzrostného vrcholu, p?i ní? se vytvá?ejí primordia květ?. III. fáze zahrnuje vznik kvě tních orgán?. IV. fáze je kvetení.     

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l^?1 of exogenous 6‐benzyl adenine (6‐BA) was administered to 3 and 7‐day‐old apple rootstocks ‘M.26’ cuttings (3 and 7 days 6‐BA) by transferring them from a rooting medium containing indole‐3‐butanoic acid to the medium containing 6‐BA. Anatomical and morphological observations revealed that the exogenous application of 6‐BA inhibited primordia formation in the 3 days 6‐BA but not the 7 days 6‐BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6‐BA than in 7 days 6‐BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6‐BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6‐BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3–7 days is the time period in which AR primordia formation occurs in cuttings of ‘M.26’ and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3–7 days period after culturing in rooting medium.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Auxins and polyamines in relation to differential in vitro root induction on microcuttings of two pear cultivars

The internal levels of indole-3-acetic acid (IAA) and polyamines (PAs) and the metabolism of indole-3-butyric acid (IBA) were studied in relation to the in vitro rooting process of two pear cultivars, the easy-to-root Conference and the difficult-to-root Doyenne d'Hiver. Doyenne d'Hiver required about a 10 times higher concentration of IBA to achieve a rooting percentage similar to that of Conference. One- or two-day exposures to IBA were sufficient to stimulate rooting but with different efficiency for each cultivar. Longer exposure to auxin strongly increased the root number in Conference, whereas root elongation was inhibited in both cultivars. The metabolism of IBA in both cultivars was not significantly different when IBA was used at a high concentration to stimulate maximal rooting in Doyenne d'Hiver. IBA was mainly conjugated into IBA glucose, which was accumulated, and a small amount was converted into free IAA in both cultivars. However, in Doyenne d'Hiver this metabolic pathway appears to be active only at a higher exogenous IBA concentration. At a high IBA concentration more callus was formed by Doyenne d'Hiver, indicating that the cells of Doyenne d'Hiver are not capable of responding to the hormone in the same manner as Conference cells. Anatomic observations indicated that the capacity to induce initial dividing cells was more efficient in Doyenne d'Hiver, but subsequently the number of root primordia formed and root development were much reduced relative to Conference. A possible correlation between these processes and an early increase followed by a decrease of free IAA was seen in Conference. By day 4, a significant increase in IAA conjugates and free putrescine was observed in Doyenne d'Hiver. This higher putrescine content may be related to the lower amount of root development. Together with previous studies these results indicate that differences in the uptake and metabolism of applied auxins may affect rooting ability and the subsequent development of adventitious roots in microcuttings of pear.Abbreviations IBA indole-3-butyric acid - IAA indole-3-acetic acid - PA(s) polyamine(s) - HPLC high pressure liquid chromatography - GC-MS gas chromatography-mass spectrometry - TCA trichloroacetic acid dansyl, 1-dimethylaminonaphthalene-5-sulfonyl - TLC thin layer chromatography - TBA terbutilic alcohol - IBAGluc IBA glucose - IAAGluc IAA glucose - IAAsp IAA aspartate     

Key phases in the formation of caveolae

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on ‘fuzzy’ charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%–62.5%) compared to free IAA or IBA applications (50.0%–33.3%), except for 2.0 mg L^–1 IBA (66.7%). However, the application of 2 mg L^–1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.     

Quantitative variations of indolyl compounds including IAA, IAA-aspartate and serotonin in walnut microcuttings during root induction

Shoots of the hybrid walnut Juglans nigra x Juglans regia contained serotonin in the micromole range and indole-3-acetic acid (IAA) in the nanomole range. The serotonin level fell by 40 % in 12 h in auxin (IBA) treated whole shoots and then reincreased to a maximum (50 %over the control) after 36 h. The same pattern was followed in the top portions of the shoot but in the shoot bases, serotonin always remained under the control level. The early decrease of serotonin was correlated with an increase in IAA-aspartate. The early decrease and peaking of the serotonin level preceded and corresponded to the increase and peaking of free IAA in the shoot bases. The initial serotonin pool in treated-to-root shoots might thus suffice for the biosynthesis of IAA and IAA-conjugated compounds. Because of its auxin-like properties, the early serotonin peak might be taken into consideration as an endogenous auxin signal for rooting in the present material. If this turns out to be so, the rooting signal for the shoot bases necessarily should come from the apices. This revised version was published online in July 2006 with corrections to the Cover Date.     

Timing of root dormancy and tolerance to root waterlogging in clonal Sitka spruce

 Actively growing root tips of Picea sitchensis (Bong.) Carr. plants are highly susceptible to damage if waterlogged, but they are known to have some tolerance after they stop growing in the autumn. This paper describes the selection of clones on the basis of root dormancy timing and the corresponding responses of their roots to over-winter waterlogging. Sitka spruce transplants of Alaska, Queen Charlotte Islands (QCI), and Washington provenances were screened for early or late root dormancy over 2 successive years. Cuttings were propagated from the selected plants and after growing on for 2 years, they were planted in transparent acrylic tubes within outdoor ‘root observation chambers’. Extension of main roots and the timing of onset of root dormancy was recorded on the clonal plants. The tubes were flooded in November and maintained with a water table 280 mm below the soil surface until March of the next year. Waterlogging caused most main root tips to die back, but within 2 months of draining regeneration occurred on the main roots below the waterlogging level. This regeneration was most commonly the growth of existing lateral tips or production of new lateral roots. Roots of early-dormant Washington plants died back on average 129 mm less than late-dormant Washington plants, and early-dormant Alaska plants had 173 mm less dieback than late-dormant Alaska plants. Differences between the clones of the QCI provenance were not significant. The 40% and 52% increases in survival depth of roots in early-dormant Washington and Alaska clones respectively indicates a potential for improving the rooting depth of Sitka spruce on seasonally waterlogged soils by planting clones selected on the basis of root dormancy. Received: 14 July 1997 / Accepted: 15 September 1997