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 gibberellin- and cytokinin-like substances in cultured shoot tissues of apple,Malus pumila cv. Jonathan,in relation to adventitious root formation

The frequencies of adventitious root formation in vitro of isolated shoots from bud cultures of apple (Malus pumila cv. Jonathan) after 1, 7 and 31 subcultures (weeks 5, 29 and 109 after the initial culture) were 5, 78 and 95% respectively. Endogenous gibberellin-like substances (GA) were extracted, chromatographed on SiO₂ partition columns, and assayed on dwarf rice (Oryza sativa cv. Tan-ginbozu). The levels of GA in shoots from the 1st, 7th and 31st subcultures were 40, 19 and 14 ng GA₃ eq./g dry weight of tissue, respectively, a trend which suggests an inverse relationship between endogenous GA level and rooting ability. This is consistent with the fact that applied GA₃ inhibits rooting in apple and many other species. The major peak of GA activity eluted coincidentally with GA₁/GA₃/GA₁₉. Endogenous cytokinin-like substances (CK) were chromatographed on paper and assayed with soybean hypocotyl sections. In contrast to the decrease in GA activity, CK activity increased 1.5–2.7 fold in the later subcultures (cytokinin activity per shoot, however, declined).     

Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

Degradation of exogenous indole-3-butyric acid and riboflavin and their influence on rooting response of papaya in vitro

Varying concentrations of riboflavin were added to a De Fossard et al. (1974) basal medium containing 10 µM IBA and the effect on adventitious root initiation on shoots of Carica papaya L. was studied. Ninety percent root initiation occurred in 11 days when 1 µM riboflavin was added to the culture medium. Smaller rooting percentages were observed and roots emerged more slowly with riboflavin concentrations greater and less than 1 µM. Tissue culture media were maintained at 27°±1°C in either darkness or 12-h photoperiods for 28 days, and concentrations of riboflavin and IBA were measured at regular intervals using HPLC analysis. In a De Fossard et al. (1974) basal medium, riboflavin concentrations (0.1, 1.0, 10.0 µM) decreased rapidly in light and were independent of the presence of IBA. IBA concentration steadily decreased when media was placed in light, and increasing riboflavin concentrations accelerated the reduction of IBA levels. Concentrations of IBA and riboflavin were stable with dark incubation.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid     

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.     

Seasonal changes in adventitious root formation in hypocotyl cuttings of Pinus sylvestris: Influence of Photoperiod during stock plant growth and of indolebutyric acid treatment of cuttings

Seedlings of Pinus sylvestris were grown for 6 weeks under natural light conditions in a temperature controlled environment room. Cuttings from these plants were rooted in tap water or in indolebutyric acid (IBA) solutions for 60 days at an irradiance of 16 W m^-2. Experiments were performed at 3-week intervals during two growth seasons. — Seasonal changes in root formation were found in control cuttings as well as in IBA treated cuttings. The number of roots and the percentage of cuttings that rooted were high during early spring and autumn. During the summer period hardly any roots were formed. Stimulation of root formation by IBA occurred manily during spring and autumn when cuttings already possessed the ability to form roots. — The influence of photoperiod during stock plant growth was also investigated. Shorter photoperiod resulted in an increase in the number of roots and rooting percentage. The period during summer where rooting was inhibited under natural light conditions was considerably shortened when stock plants were grown at a photoperiod of only 4 h. The results demonstrate the importance of the growing conditions for stock plants for subsequent root formation. The results are discussed with special reference to the role of irradiance.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

Effect of sucrose on adventitious root regeneration in apple

We have examined the effect of sucrose on adventitious root formation in apple microcuttings and in 1-mm stem slices cut from apple microcuttings. The sucrose concentration influenced the number of adventitious roots, but at a broad range of sucrose concentrations (1–9%) the effect was small. In addition, there was an interaction between sucrose and auxin: increasing the sucrose concentration shifted the dose–response curve of auxin to the right. When slices were cultured on medium without sucrose for the initial period (0–48 h), rooting was reduced whereas 48-h culture without application of sucrose had hardly any effect or even a slight promotive effect in a later period (48–120 h). The results show that during adventitious root formation, applied sucrose is used as a source of energy and building blocks but they are also in accordance with a possible regulatory role of sucrose.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Adventitious root formation 'in vitro' in apple rootstocks (Malus pumila) I. Factors affecting the length of the auxin-sensitive phase in M.9

The length of the auxin-sensitive phase of root initiation 'in vitro' in the apple rootstock M.9 ( Malus pumila Mill.) has been determined using the auxins indol-3yl-acettc acid (IAA) at 2.8 × 10⁻⁵ M and indol-3yl-butyric acid (IBA) at 1.5 × 10⁻⁵ M in the presence and absence of 10⁻³ M phloroglucinol (PG). PG synergised IBA-induced rooting after 4 days exposure, but contact times exceeding 8 days decreased root number. In contrast, PG consistently synergised IAA-induced rooting in the dark for contact periods up to 13 days with the highest rooting being recorded at 9 days. An irradiance of 20 W m⁻² from fluorescent lamps halved IAA-induced rooting irrespective of the presence or absence of PG. The culture of shoots at temperatures of 22,25 and 29°C during the root initiation phase (auxin present) and the root emergence phase (auxin absent) produced no difference in rooting response. In the presence of PG the use of liquid culture in place of agar-solidified culture during the auxin-sensitive phase reduced root number but not rooting percentage.     

Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings

Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca²⁺]_cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca²⁺] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.     

A strigolactone signal is required for adventitious root formation in rice

Background and Aims Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulating root development. This study examines whether SLs play a role in mediating production of adventious roots (ARs) in rice (Oryza sativa), and also investigates possible interactions between SLs and auxin.Methods Wild-type (WT), SL-deficient (d10) and SL-insensitive (d3) rice mutants were used to investigate AR development in an auxin-distribution experiment that considered DR5::GUS activity, [³H] indole-3-acetic acid (IAA) transport, and associated expression of auxin transporter genes. The effects of exogenous application of GR24 (a synthetic SL analogue), NAA (α-naphthylacetic acid, exogenous auxin) and NPA (N-1-naphthylphalamic acid, a polar auxin transport inhibitor) on rice AR development in seedlings were investigated.Key Results The rice d mutants with impaired SL biosynthesis and signalling exhibited reduced AR production compared with the WT. Application of GR24 increased the number of ARs and average AR number per tiller in d10, but not in d3. These results indicate that rice AR production is positively regulated by SLs. Higher endogenous IAA concentration, stronger expression of DR5::GUS and higher [³H] IAA activity were found in the d mutants. Exogenous GR24 application decreased the expression of DR5::GUS, probably indicating that SLs modulate AR formation by inhibiting polar auxin transport. The WT and the d10 and d3 mutants had similar expression of DR5::GUS regardless of exogenous application of NAA or NPA; however, AR number was greater in the WT than in the d mutants.Conclusions The results suggest that AR formation is positively regulated by SLs via the D3 response pathway. The positive effect of NAA application and the opposite effect of NPA application on AR number of WT plants also suggests the importance of auxin for AR formation, but the interaction between auxin and SLs is complex.     

Light modulates the root tip excision induced lateral root formation in tomato

During plant growth and development, root tip performs multifarious functions integrating diverse external and internal stimuli to regulate root elongation and architecture. It is believed that a signal originating from root tip inhibits lateral root formation (LRF). The excision of root tip induced LRF in tomato seedlings associated with accumulation of auxin in pericycle founder cells. The excision of cotyledons slightly reduced LRF, whereas severing shoot from root completely abolished LRF. Exogenous ethylene application did not alter LRF. The response was modulated by light with higher LRF in seedlings exposed to light. Our results indicate that light plays a role in LRF in seedlings by likely modulating shoot derived auxin.     

The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of York and Vermont Spur Delicious apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K⁺, Mg⁺⁺ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K⁺, 1.5 and 3.0 mM Mg⁺⁺, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K⁺ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.     

Reduced glutathione promotes callus growth and shoot development in a shoot tip culture of apple root stock M26

Reduced glutathione (GSH) was applied to prevent browning in shoot tip explants of apple (Malus pumila Mill.). Development was compared between shoot tips treated either by (a) dipping into 0.1 mm GSH solution prior to culture (dip treatment), (b) dipping into the GSH solution and transferring to a medium containing 0.1 mm GSH (dip-and-add treatment), or (c) without dipping or culturing with GSH (control). In the dip treatment, 100% of shoot tips developed into normal shoots after 120 days, while the results with the dip-and-add treatment and control were 50 and 40%, respectively. The results show that application of antioxidant GSH in the initial phase of culture promoted the normal development of shoot tips. Received: 10 October 1997 / Revision received: 12 January 1998 / Accepted: 24 January 1998     

Histological study of initiation and development in vitro of adventitious roots in minicuttings of apple rootstocks of M 26 and EMLA 9

Apple rootstocks M 26 and EMLA 9 'COST' shoots propagated in vitro were used for the histological study of initiation and development of adventitious roots after a brief induction pretreatment. The results show that there are differences in mode and timing of initiation and development of adventitious roots between the two varieties. In M 26, adventitious roots were directly initiated from the derivatives of the cambium, some of which were immediately transformed into meristemoids in situ 36 h after pretreatment. Subsequently, meristemoids differentiated into root primordia. Development of adventitious roots were completed when they emerged at the surface of stem bases 10 days after pretreatment. In EMLA 9, before the meristemoids formed, internal cell files were formed by continuous divisions of cambial cells. The cells were regularly arranged in files external to the cambium. On the fourth day after pretreatment, some cells in the outermost layers of these files became meristematic, started to divide and turned into meristemoids, which differentiated into root primordia. The cells of the files between the root primordium and the cambium were transformed into vascular tissues which connected the vascular systems of the adventitious roots and stems.     

Effect of light on vanillin precursors formation by in vitro cultures of Vanilla planifolia

A cluster culture of Vanilla planifolia Andr. has been established from shoots. It has been maintained in the dark. Various light conditions had little effect on its growth. However, the light conditions did affect the production in both quantity and quality of compounds associated with the vanillin pathway, particularly the 4-hydroxy-3-methoxybenzyl alcohol.Abbreviations NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)-purine - BA 6-benzylaminopurine     

Interaction of explant type and indole-3-butyric acid during rooting in vitro in a range of difficult and easy-to-root woody plants

Axillary shoots of woody species that were easy-to-root (Forsythia × intermedia, Betula pendula and juvenile Quercus robur) and difficult-to-root (Syringa vulgaris, ‘intermediate’ phase Q. robur and Daphne cneorum), were used to obtain various explant types (Entire, Detipped, Nodal or Internodal) that differed in their rooting responses. When apices or axillary buds were removed from explants, rooting levels decreased, and the time to root and loss of synchrony of rooting increased. Exogenous indole-3-butyric acid generally overcame these trends, and was essential for root induction in ‘intermediate’ Quercus and Daphne, and in all internode explants. The interaction between explant type and indole-3-butyric acid suggests a role for endogenous factors in the initiation and development of rooting. The internode explant has potential as a tool for further physiological and biochemical studies, as it is auxin-dependent and provides rooting-permissive or non-permissive states. This revised version was published online in June 2006 with corrections to the Cover Date.