The relationship between rooting of poplar shoots and ATPase activity of their crude microsomal vesicles

Poplar shoots raised in vitro were induced to root by a 7 h passage on an auxin (1-naphthaleneacetic acid) medium. The percentage of rooting was reduced from ± 97% to ± 47% when vanadate (200 µM) was included in the auxin medium. Introduction of vanadate in the medium without auxin after the 7 h induction on auxin medium, did not inhibit rooting but affected only the development of the roots produced. The Mg²⁺-dependent ATPase activity of the microsomal vesicles of poplar shoots was increased after 7 h induction on rooting medium and corresponded to an increase in the Vmax of the enzyme. Results from experiments using some inhibitors of the polyamine metabolism suggested that this pathway was not involved in the increase of this activity. The auxin had no effect on the in vitro ATPase activity at any concentration tested except at about 2 mM where it was inhibitory, probably due to a change in the conformation of the enzyme. The transient increase of indole-3-acetic acid during rooting induction could be responsible for the increase in the level of the enzyme. The inhibition of root formation and growth by vanadate indicates strongly that the ATPase activity may be necessary for the induction and expression of rooting.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.     

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.     

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.     

Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Promotion by phloroglucinol of adventitious root formation in micropropagated shoots of adult wild cherry (Prunus avium L.)

Micropropagated shoots of wild cherry (Prunus avium L.) produced roots in auxin-free medium. Phloroglucinol (PG) increased the proportion of shoots that rooted, while phloretic acid reduced this response in medium with or without PG, and cancelled the promotive effect of PG. Concentration of PG also significantly affected rooting in media with and without auxin. The proportion of shoots rooting in media containing auxin, or auxin plus PG, increased with the number of successive subculture, but the proportion that rooted with PG alone was unaffected by the number of subcultures. Before the shoots had become responsive to auxin, 1 mM PG was more effective than auxin in inducing root formation.     

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.     

Rooting of blue honeysuckle microshoots

Rooting of axillary shoots of two blue honeysuckle forms, Lonicera caerulea f. caerulea and L. caerulea f. edulis, was studied. Both in vitro and ex vitro rooting procedures were used, and the effects of mineral and auxin concentrations of the rooting media were tested. Reduced mineral nutrient concentrations of modified MS medium allowed more root elongation but did not affect the primary root number. The rooting percentage was high (≥ 90) in the form caerulea microcuttings but low (< 40) in the form edulis microcuttings when not treated with auxin. The rooting frequency and primary root number of the form edulis shoots could be increased up to 100 with 10 roots per microcutting, in the continuous presence of auxin. The continuous auxin treatments repressed the elongation and increased the diameter of primary roots and induced callus formation at the base of the shoots. Differences in root systems were related to equimolar concentrations of the auxins indole-3-butyric acid, indole-3-acetic acid and α-naphthaleneacetic acid, but the differences were diminished after one month ex vitro. After transfer ex vitro, several of the roots formed in vitro and some microcuttings died. A high rooting percentage and a good ex vitro survival and root growth of the form edulis microplants were achieved by a 7-day pulse with 4 μM indole-3-butyric acid followed by rooting ex vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

植物生长调节剂对黑木相思优树腋芽增殖及生根的影响

为建立黑木相思(Acacia melanoxylon)快繁技术体系,以含1个腋芽的无菌茎段为材料,研究了植物生长调节剂对其增殖和生根的影响。结果表明,6-BA 极易诱导黑木相思愈伤组织形成,但芽长势较差,不利于腋芽增殖体系的建立。而生长素既能诱导黑木相思生根,又能诱导腋芽增殖;将无菌茎段接入MS+IAA 0.5 mg L^-1+IBA 0.5 mg L^-1培养基中培养20 d的生根率为98.41%,培养40 d的腋芽增殖倍数为2.36,单株繁殖系数为6.57。这是首次成功建立高效、简便的生根和增殖同步发生的黑木相思直接器官发生途径的组培技术体系。     

In vitro rooting of almond (Prunus dulcis Mill.)

Summary Shoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.     

Micropropagation of juvenile and adult Quercus suber L.

This paper describes research on the application of tissue culture techniques to the micropropagation of cork oak (Quercus suber L.), a forest species of ecological and industrial importance in the Mediterranean area. Apical buds and nodal stem segments were employed as initial explants. Their origins were young seedlings, stump sprouts and sprouts formed on cuttings collected from old trees.The action of the mineral medium and growth regulators was studied in the multiplication stage. Media with low concentrations of ions, such as Sommer's or Heller's, are more suitable for growth and proliferation of explants than other media richer in salts. It was also observed that cytokinin (BA) must be present for the culture development. Adding low concentrations of auxin (NAA) to the medium improves the multiplication rate, especially in vegetative material of adult origin.The auxin type is the most important factor in the promotion of rhizogenesis. The method of application determines the quality of the root system. Treatment with low concentrations of IBA added to the rooting medium gives the best results.High sucrose concentration also improves rooting. Diluting the mineral rooting medium is slightly favourable, although there is no significant difference between it and the standard mineral concentration.Abbreviations D Durzan's - GD Gresshoff & Doy's - H Heller's - L Lepoivre's - MS Murashige & Skoog's - SH Schenk & Hildebrandt's - S Sommer's medium     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid     

Glutathione and glutathione disulfide affect adventitious root formation and growth in tomato seedling cuttings

To study the relationship between glutathione and rooting, tomato seedling cuttings, grown on basal- or on auxin-supplemented media, were treated with the reduced (GSH) or oxidized (GSSG) form of this antioxidant. In turn, the consequences of the depletion of GSH pool on rooting were tested using l-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis. Effects of the aforementioned treatments on rooting response were assessed. GSH treatment promoted root formation on cuttings grown on both basal- and auxin-supplemented media. Whereas GSSG did not affect the number of roots formed by cuttings grown on basal medium, it strongly enhanced the rooting stimulatory effect of auxin treatment. GSH depletion resulting from BSO application did not change the number of roots formed. All the tested compounds, namely GSH, GSSG, BSO and auxin, had a strong inhibitory effect on the elongation of regenerated roots. Supplementing the rooting medium with glutathione efficiently increased the GSH level in the rooting zones, while addition of BSO led to a strong decrease in endogenous GSH level. Neither of the treatments affected the level of GSSG. Exogenous auxin affect neither GSH nor GSSG levels in rooting zones; however, in the regenerated roots, GSH level was significantly higher when the organs were formed on auxin-supplemented medium. Patterns of GSH distribution in the roots regenerated on basal- and auxin-enriched media were studied using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. GSH was found in the root apical meristem and in the elongation zone. Auxin did not change the GSH distribution; however, the number of fluorescent cells was higher when roots were regenerated on auxin-supplemented medium.     

Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild-type and indoleacetic acid (IAA)-overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA-overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster . No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.     

Improved shoot development and rooting from mature cotyledons of sunflower

Regeneration and development of shoots from sunflower cotyledons were improved by optimizing explant age, plant growth regulator concentrations, and duration of exposure to plant growth regulators during shoot initiation, development and rooting. Shoot initiation required only a brief exposure to auxin and cytokinin, and minimizing the duration of exposure to high levels of plant growth regulators improved shoot development. Rooting was improved in terms of both the number of shoots that rooted and the time required for rooting by incorporating activated charcoal in the lower layer of a 2-layer rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of culture media for in vitro rooting of Malus domestica Borkh. cv. Compact Spartan

Shoots of Malus domestica Borkh. cv. Compact Spartan raised in vitro do not root in a single auxin medium. Components of the rooting medium were tested not only for root initiation but also for root elongation. Root emergence and further growth were inhibited by a too prolonged auxin treatment, the presence of NH4NO3 and the lack of substrate aeration. Saccharose was essential to achieve highly reproducible root growth on agar but was not necessary on watered vermiculite. Ca(NO3)2 stimulated root initiation, emergence and growth and improved their viability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal Changes in Auxin Effects on Rooting of Stem Cuttings of Populus nigra and its Relationship with Mobilization of Starch

Stem cuttings of Populus nigra were treated with 10 and 100 mg/1 each of IAA., IBA, 2,4-D and NAA at one month intervals and observations were recorded for the morphophysiological status of the branches, their starch content and their rooting response. — The first phase characterized by delayed, short and scarce roots and the high starch content of cuttings coincided with the onset of winter dormancy in November lasting through February. It was followed by a phase of vigorous rooting and low starch content of cuttings coinciding with the renovation of growth activity in February lasting through October, except in April and May when rooting was more or less completely nullified. — The poor rooting in winter was caused by low activity of hydrolyzing enzymes not mobilizing starch into soluble sugars; and profuse rooting during active growth period by high activity of hydrolyzing enzymes caused by endogenous auxin, resulting in mobilization of reserved food materials necessary for the initiation and development of roots. The low rooting in April and May is ascribed to the fact that bulk of the mobilized food was used up in the growth of sprouted branches leaving very little for rooting when these cuttings were planted. — The seasonal changes in the effectiveness of exogenously applied auxins also appear to be related with the level of endogenous auxin. In June endogenous auxin was high due to high meristematic activity, the exogenously applied auxins raising it to supra-optimal levels that were inhibitory. On the other hand, in October exogenously applied auxins enhanced rooting by raising it to an optimal level as the production of endogenous auxin had been decreasing gradually due to lowering temperatures. — The results demonstrate that auxin effect on differential rooting with season in this plant is determined by the physio-morphological status of the branches that govern the production of endogenous auxin and is mediated primarily through its effect on mobilization of reserve food materials caused by enhanced activity of hydrolytic enzymes.     

In vitro rooting of cloned shoots in Pinus pinaster

In vitro plantlet regeneration will be a sueful tool in reforestation only when rooting problems are overcome. Our study was therefore focused on Pinus pinaster rooting conditions.
Methods were used which permit multiple axillary (instead of adventitious) bud production in order to obtain genetically identical explants and hence to procure more homogeneous resutls. The hormonal treatment efficiency was then tested on several clones for rooting. Different substrates were compared. A mixture of peat and perlite (1/3, v/v) permitted rooting and the further development of the root system if humidity was properly controlled. The use of an agar medium avoided this difficulty; it supported root induction but resulted in limited growth.
For hormonal treatment, explants, submitted to a 12-day-period with NAA (10⁻⁶ M ) and then subcultured in an auxin-free medium, gave rooting percentages ranging from 80 to 100%. In terms of rooting quality (intensity, growth rate and branching ability), a 15-to 19-day-period of auxin treatment was more effective.
Application of this optimal treatment to several clones revealed slight differences within clones and between clones (80 to 100% of rooting according to the clone). Regenerated plantlets were able to undergo an autotrophic growth after transplantation under greenhouse conditions.