Isolation, Purification, and Characterization of an Endogenous Root-promoting Factor Obtained from Basal Sections of Pear Hardwood Cuttings

Basal segments taken from Old Home and Bartlett pear hardwood cuttings collected at intervals during the rooting period in September were extracted with ethanol and fractionated by paper chromatography in different solvent systems. Different zones on the chromatograms were bioassayed by the mung bean rooting test, which showed high levels of promotion in Old Home basal extracts when the cuttings were obtained during the period of maximum rooting. Extracts from Bartlett cuttings, however, showed considerably less promotion activity in the bioassay but did show high levels of inhibitory activity.

After the easily-rooted Old Home cuttings had been in the rooting medium for 10 days, a highly active endogenous root-promoting material was found in extracts from basal segments of cuttings having buds and which had been treated with indolebutyric acid. Similar extracts obtained from disbudded cuttings, or from cuttings with buds but not treated with indolebutyric acid, lacked this rooting-factor. Extracts obtained from all types of the difficult-to-root Bartlett cuttings also lacked this rooting-factor. The latter is believed to be produced by physiologically active Old Home buds, and is very effective in the mung bean bioassay, even at extremely low concentrations.

From paper chromatographic studies, tests with spray reagents, solubility determinations, biological tests, UV spectrum analysis, and infrared spectroscopy, it is believed that this rooting factor could be a condensation product between exogenous auxin (indolebutyric acid) and a phenolic compound produced by physiologically active Old Home pear buds.

     

Effects of autoclaving and charcoal on root-promoting substances present in water extracts made from gelling agents

The root-promoting ability of water extracts made from gelling agents (agar and Gelrite) was investigated using the mungbean rooting bioassay. Autoclaving these water extracts decreased the number of roots in mungbean cuttings compared to the controls. The addition of activated charcoal to the water extracts from Agar Bacteriological and Agar Commercial Gel had no effect on their root-promoting ability. Extracts with exogenous indole-3-butyric acid (IBA) which were treated by autoclaving or via a freeze-thaw cycle, significantly increased rooting. However, incorporation of activated charcoal to similar IBA-containing extracts reduced rooting. Our results indicate that more attention should be given to the choice of gelling agent and its interaction with other additives in the media used during tissue culture.     

N-phenyl indolyl-3-biityraitiide and phenyl indole-3-thiolobutyrate enhance adventitious root primordium development

N-phenyl indoryl-3-butyramide (NP-IBA) and phenyl indole-3-thiolobutyrate (P-ITB) are new synthetic auxins that enhance rooting of bean ( Phaseolus vulgaris cv. Top Crop) hypocotyl cuttings and jack pine ( Pinus banksiana Lamb.) seedling cuttings. In comparison with an equal concentration of indole-3-butyric acid (IBA), NP-IBA treatment resulted in greater rooting of bean and jack pine seedling cuttings. IBA and P-ITB treatment at equal concentration were equally effective in inducing rooting of bean cuttings. However, in comparison with an equal concentration of IBA, P-ITB treatment promoted greater rooting of jack pine seedling cuttings, but P-ITB treatment was less effective than NP-IBA treatment. Application of crystalline chemical to jack pine seedling cuttings indicated lesser toxic effects and earlier and greater root-promoting effects of P-ITB and NP-IBA treatments, in comparison with IBA treatment. The earlier rooting of NP-IBA-treated jack pine seedling cuttings was positively related to increase in fresh weight of basal 1 cm stem before primordia were initiated. Weight of basal steins of NP-IBA-treated cuttings initially increased during propagation at day 2 in comparison with the control, and at day 3 in comparison with IBA-treated cuttings. Results that were obtained with seedling jack pine cuttings were generally confirmed for cuttings from 7-year-old trees.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Rooting Substances in Water Extracts of Castanea sativa and Salix viminalis

The rooting activity of water extracts of cuttings of chestnut (Castanea sativa Mill.) and willow (Salix viminalis L.) was studied throughout the year by the bean rooting test. The chestnut extract nullified the rooting activity of 10^?5M IAA, but on the whole it did not modify the rooting pattern of the bean cuttings. The willow extract increased rooting and showed a rhythm in the content of endogenous growth substances. IAA was found at the time when the extract showed the strongest rooting activity. The extract from willow contained two synergistic root-promoting fractions with the R_f's of 0.0–0.2 and 0.6–0.8 in isopropanol: ammonia: water. The latter one promoted rooting without added IAA. In this fraction a high amount of catechol was detected. No clear correlation was found between the ability to induce roots and the total phenol content in the two species.     

The Relationship Between Rooting of Dahlia Cuttings and the Presence and Type of Bud

Dahlia cuttings with actively growing buds are relatively hard to root as compared with those having non-growing or inhibited buds. In cuttings containing buds which sprouted during the rooting period, an inverse relationship was found between rooting percentage and growth rate of buds. Reproductive buds suppress rooting more than vegetative ones. Removing the growing terminal, parts of the cuttings (vegetative or reproductive) increased rooting percentage of cuttings. It is suggested that growing buds may affect rooting of cuttings in two opposed directions. The first is inhibition of rooting by competing with the roots for metabolites and the second is promotion of rooting by enhancing cambial activity.     

Vegetative Propagation of Pinus sylvestris

Methods for the vegetative propagation of Pinus sylvestris L. from interfascicular shoots are described. Using 5-year-old plants the outgrowth of interfascicular shoots was promoted by removal of terminal and lateral buds; this response was augmented substantially by application of cytokinin, tri-iodobenzoic acid, alar and morphactin alone and especially in combination. The rooting capacity of shoot cuttings from interfascicular shoots appeared to be largely determined by the state of growth of the stock plant. Cuttings from dormant stock plants subjected to short-day treatment followed by a period of low temperature gave the best rooting, especially when the cuttings themselves had been cold-stored prior to planting. Rooting was optimal when such cuttings were treated with a mixture of 25 mg/l of indolebutyric acid and 25 mg/l of napthaleneacetic acid as a 48 h basal soak, and were planted on a heated mistbench under extended illumination; over 90% of such cuttings could be rooted. These results are discussed in relation to bud activity, endogenous hormone levels and promoting tissue extracts also tested.     

Treatment of bean cuttings by chestnut extracts during rooting modifies their IAA-oxidase activity

The IAA-oxidase activity in bean cuttings ( Phaseolus vulgaris L. cv. Contender) treated with extracts from juvenile and adult chestnut ( Castanea sativa Mill.) was studied in the light of the effect of the extracts on the rooting activity of some auxins. Extracts from adult chestnut increased IAA-oxidase activity in bean cuttings. Extracts from juvenile chestnut inhibited IAA-oxidase activity only slightly, but substantially reduced the IAA-oxidase activity of bean cuttings pre-treated with adult chestnut extracts. These findings provide evidence that there is a relationship between the IAA-oxidase system and the effect of chestnut extracts on IAA-induced rhizogenesis in bean cuttings.     

Root-promoting Substances in Salix alba

Root-promoting substances were extracted from softwood cuttings of Salix alba L. by centrifuging them with water or by shaking the ground freeze-dried stems with water. Rooting substances were partitioned by paper chromatography or chemical fractionation and their rooting activity was tested by mung bean cuttings. Both extracts indicated three major root -promoting fractions at Rf 0-0.1, 0.7-0.8, and 0.3-0.4 in a decreasing order of their activities when paper chromatographed with isopropanol:ammonia:water 8:1:1 v/v. The strongest one indicated an apparent synergistic rooting effect with indol-3yl-acetic acid (IAA) regardless of the extraction method. These results indicate that water can extract from freeze -dried sample the similar rooting substances found in the centrifugal diffusates. The Rf 0–0.1 fraction consisted of at least four fractions and the strongest one did not move from the starting line on the chromatogram when isopropanol:ammonia:water 8:1:1 was used. This starting line fraction was extremely strong in rooting activity and its highest concentration resulted in 8.7 times as many roots as controls. More thain additive rooting effect between IAA and the fraction was found only at the highest concentration. The fraction was very soluble in water but insoluble in chloroform or ethyl ether and only stimulated rooting of mung bean cuttings when it was applied within 3 days after cuttings were made. It had no effect in lengthening roots. The starting line fraction was further found to have four root-promoting subfractions at Rf 0.05, 0.35, 0.65, and 0.85 when it was chromatographed in 60 % isopropanol. Among these four, the subfractions at Rf 0.65 and 0.35 were strongly root promotive and displayed more than additive root promotion with IAA at the highest concentrations studied.     

Effects of Extracellular Extracts from Leaves on the Tuberization of Cuttings of Potato (Solanum tuberosum L.)

Extracellular extracts from leaves were applied to moderately induced cuttings of potato. The more inductive cycles the plants from which the leaf extracts were obtained had experienced, the lower the percentages of tuberization in thcuttings and the lower the tuber yields on the central buds.     

萘乙酸对阳春砂不同插穗扦插繁殖效果的影响

以阳春砂根状茎茎尖、根状茎、直立茎芦头3种不同材料为插穗,研究不同浓度的ɑ-萘乙酸(NAA)对扦插效果的影响,以筛选出最佳的NAA处理浓度,寻找除分株繁殖外的高效营养繁殖方法。结果表明,未经NAA处理的根状茎茎尖、根状茎和直立茎芦头3种插穗的出芽率和生根率比较低,出芽率依次为10.0%、3.3%和23.3%;生根率依次为23.3%、10.0%和3.3%。而200 mg·L-1的NAA处理可以显著提高根状茎茎尖、根状茎和直立茎芦头3种插穗的存活率、出芽率和生根率;与对照比较,3种插穗的存活率依次提高17.7%、23.4%和26.7%;出芽率依次提高13.3%、50.3%和53.3%;生根率依次提高43.3%、26.7%和53.4%。200 mg·L-1的NAA处理可以显著提高3种插穗再生根和再生芽的生长指标。根状茎茎尖再生根的数量、长度、直径分别比对照提高56.7%、82.6%和29.2%;根状茎的再生根长度显著比对照长203.5%;直立茎芦头的再生芽的长度和直径比对照提高25.2%和35.2%。NAA处理后的根状茎茎尖插穗的芽再生方式有两种,一种是原来的茎尖继续生长,另一种是从插穗的节上分化出新芽。阳春砂根状茎茎尖、根状茎和直立茎芦头3种插穗经200 mg·L-1NAA处理后可以显著提高成活率,这为阳春砂种苗生产建立了分株繁殖以外的另一种高繁殖系数的营养繁殖方法。     

Explant Orientation and Polarity Determine the Morphogenic Response of Epicotyl Segments of Troyer Citrange

The morphogenic pathway of adventitious bud and shoot regenerationat the ends of Troyer citrange epicotyl cuttings is determinedby polarity and explant orientation. In explants planted verticallywith the basal end inserted in the medium, bud formation atthe apical end occurs by direct organogenesis. Bud growth andsubsequent shoot formation is markedly increased by the additionof 6-benzyladenine (BA) to the medium. This growth regulatoralso increases the number of buds formed. When they come intocontact with the culture medium, both the apical end and thebasal end of the cuttings form a vigorous callus with many xyllaryelements, more numerous in the calli from the basal end. Inthese calli, buds differentiate by a process of indirect organogenesis.This indirect regeneration pathway requires the addition of6-benzyladenine to the medium, and the number of buds formedis higher at the apical end than at the basal end of the cuttings.This pathway of regeneration is reduced as the position of thecuttings during incubation deviates from the normal uprightvertical position. Thus, for the basal end of the cuttings,the number of buds and shoots formed is higher when the explantsare placed vertically than when they lie on the surface of themedium. For the apical end, this number is higher in explantsplaced horizontally than when inserted vertically in the mediumin an inverted position. Copyright 1999 Annals of Botany Company Troyer citrange, Citrus sinensis x Poncirus trifoliata, explant orientation, histology, hormone dependence, morphogenesis, organogenesis, polarity, xylogenesis.     

Floral-stimulus activity in tobacco stem pieces

The growth patterns of axillary buds of dayneutral tobacco (Nicotiana tabacum L. cv. Wisconsin 38) plants were assessed by using expiants of single buds attached to leafless stem cuttings and allowing the buds to grow to flowering without additional manipulation. Buds located 5, 10 and 15 nodes below the inflorescence were employed. For a given bud position, when a cutting had few internodes the growth pattern of a bud tended to fall into one of two groups: buds that produced few-noded shoots and buds that produced many-noded shoots. For example, in a group of 13 cuttings composed of bud 5 with 2 associated internodes, 11 buds produced 14.2 nodes (range, 11–17) and 2 buds produced 32.0 nodes (range, 30–34). As the number of internodes on the cutting increased, the number of buds producing few-noded shoots increased and the number of nodes produced decreased (e.g. in contrast to the data above, all 5th buds with 6 internodes produced 12.8 nodes; range 11–15). When cuttings from the 3 positions had the same number of internodes, the more apical cuttings had buds that produced fewer nodes (e.g. for cuttings with 6 internodes all 5th buds produced 12.8 nodes, all 10th buds produced 15.5 nodes and 85% of 15th buds produced few-noded shoots with 19.3 nodes). The number of nodes produced by a bud was a function of the original position of the stem piece and not the original position of the bud. That is, bud 5 associated with the 6 internodes below it produced 12.8 nodes and bud 10 associated with essentially the same 6 internodes (i.e. the 6 above it) produced 12.9 nodes while bud 10 associated with the 6 internodes below it produced 15.5 nodes. Thus, the number of nodes produced by a bud was dependent upon the original main-axis position of the cutting as well as the number of internodes on the cutting. Buds forced to grow out in situ on main axes devoid of leaves produced substantially more nodes than similar buds on cuttings. Buds isolated without associated internodes produced many-noded plants with a number of nodes similar to that of plants grown from seed. The simplest interpretation of these data is that stem pieces contain floral-stimulus activity and that this activity is present in a gradient with the highest activity being located in the apical part of the stem.We thank Susan Smith and Harry Roy (Rensselaer) for comments, and the National Science Foundation for financial support (IBN-9003739 to C.N.M.).     

Removal of the stem terminal and application of auxin change carbohydrates in Pinus banksiana cuttings during propagation

This study determined how surgical removal of the stem terminal, with indole-3-butyric acid (IBA) treatment, influenced concentrations and partitioning of carbohydrates in Pinus banksiana Lamb, cuttings during propagation. Seedlings and cuttings that originated from 90-day-old stock plants were untreated or treated by removing the stem terminal, followed by application of IBA to the severed apical or basal (cuttings only) stem. Fresh and dry weights of the basal 1-cm stems of cuttings were determined daily for the first 10 days of propagation (i.e., before roots were visible). In addition, basal 1-cm stems, upper (ca 9-cm) stems and needles of seedlings and cuttings were analyzed for sucrose, soluble reducing sugar and total non-structural carbohydrate. Net concentrations of each carbohydrate in cuttings were obtained by subtracting corresponding concentrations for similarly treated seedlings, yielding data directly related to only the physiology of rooting. Data for cuttings indicated that presence of the stem terminal combined with applied IBA positively influenced rooting through processes that increased basal stem fresh and dry weights before root emergence. Removal of the stem terminal influenced accumulation of net total carbohydrate in cuttings, but the major effect was on carbohydrate partitioning. Either type of IBA treatment after removal of the stem terminal usually resulted in different net carbohydrate concentrations in each tissue source of cuttings, compared with only removal of the terminal. Neither basal nor apical IBA treatment of cuttings without stem terminals yielded results for carbohydrate accumulation and partitioning like those obtained with intact cuttings. Removal of the stem terminal, even if followed by IBA treatment, may have lessened rooting potential of cuttings because it resulted in greater reducing sugarstarch concentration ratios in basal stems compared with those in intact cuttings.     

The Nature of Root-Promoting Substances in Lycopersicon esculentum Seedlings

Determination of the organ sites and chemical nature of root-promoting substances in Lycopersicon esculentum seedlings was made. Results of organ excision indicated that the young cotyledons were the primary site of root-promoting substances. As the cotyledon matured, its root-promoting activity decreased while that of the developing plumular leaves increased. Application of 2,3,5-triiodobenzoic acid (TIBA) at 10^?3 M in lanolin to the hypocotyl below the cotyledonary node significantly prevented root formation. Bioassay, using a new tomato rooting test, of the basic, acidic and bound fractions of the cotyledon extracts after paper chromatography showed biological activities in all three fractions. Gas-liquid chromatography of selected active regions of the paper chromatogram resulted in a partial tentative identification of indolepropionic acid, 5-methylindole, 5-hydroxy-indole and 3-indoleacetonitrile. The combined evidence indicated that the root-promoting substances in tomato seedlings resemble auxin compounds.     

Sequential rooting media and rooting capacity of Sequoiadendron giganteum in vitro. Peroxidase activity as a marker

The rooting capacities of tips of seedling, juvenile and mature shoots of Sequoiadendron giganteum were compared on different rooting media (inductive and expressive media) after passage on an elongating medium. None of the cuttings rooted when continuously kept on medium containing the auxin NAA and vitamin D2. Peroxidase activity of all those cuttings on NAA+D2 first increased during the 7–9 first days and decreased in the days after. Rooting was obtained by transfer of the cuttings after periods longer than 7–9 days from the NAA+D2 inductive medium to a basal medium supplemented or not with rutin (expressive medium). The rooting capacity was emphasized by rutin treatment and was in correlation with the peroxidase peak reached on the NAA+D2 medium. Seedlings, characterised by the highest peroxidase activity, were most performing in rooting.Abbreviations BM basal medium - D2 ergocalciferol - NAA naphtaleneacetic acid     

Flower bud differentiation in Salix pentandra as affected by photoperiod, temperature and growth regulators

Flower bud initiation in seedlings and vegetatively propagated plants of Salix pentandra from different locations has been studied under controlled conditions. In mature plants flower bud formation was induced by 2-chloroethyltrimethylammoniumchloride (CCC) and by short day treatment. The effect of CCC was antagonized by GA₃. The critical photoperiod for flower bud formation was about 18 h for a southern clone (from 49°48'N), but cuttings of a northern ecotype (from 69°39'N) formed flower buds even at 24 h photoperiod. Generally, flower bud formation occurred simultaneously with apical growth cessation. However, apical growth cessation was not a prerequisite for floral initiation and flower buds were also found in elongating plants. Seedlings up to 60 days old did not form flower buds in growth chamber studies. The length of the juvenile phase has not been studied in detail, but cuttings taken from seedlings approximately 20 cm high and 60 days old were able to develop flower buds when treated with CCC. A gradual transition from the juvenile to the mature phase was obtained by repeated pruning of seedlings grown at 18°C and 24 h photoperiod.     

Auxin in scapes,flower buds,flowers, and fruits of daffodil (Narcissus pseudonarcissus L.)

Summary Diffusates from flower buds, flower fruits, and scape segments, and extracts of flower stalks of Narcissus pseudonarcissus contain an auxin active in the Avena geo-curvature test. The auxin behaved like indole-3-acetic acid (IAA) in thin-layer chromatography (TLC) with neutral and basic solvents on different adsorbents. After TLC, the auxin of the extracts showed chromogenic reactions identical with those of IAA; in gas-liquid chromatography on two different columns, the purified substance, after methylation, appeared at the retention time of IAA methyl ester. The auxin content of the extracts has been estimated to be equivalent to ca. 10 g IAA kg^–1 fresh weight. Diffusates, collected at the basal end of excised flowering apices and of scape segments at different developmental stages, showed highest auxin activity when collected from old buds and young flowers, and from the basal, rapidly elongating scape regions. The diffusible auxin obtained from scape segments was very likely produced by the segments themselves. Thus, the shoot of Narcissus appears to possess two different sites of auxin production, namely, the apical region represented by the flower bud, the flower or the fruit, and the scape.Abbreviations IAA indole-3-acetic acid - IAA-OMe indole-3-acetic-acid methyl ester - TLC thin-layer chromatography - GLC gas-liquid chromatography     

Compression wood in rooted cuttings of Douglas-fir

Rooted cuttings of three clones of Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco] were growing plagiotropically two seasons after propagation. The oblique growth habit of cuttings was due, in part, to dorsoconvex curvature of the wood of the original 12-cm cuttings after rooting. In cross sections taken 1.5–2.0 cm from the bases of cutting stems, the total widths of xylem and compression wood were significantly greater on the morphologically upper (convex) than on the lower sides of the stems. This was due to an asymmetry in the growth ring produced the season following rooting. In nearly every cross section examined, 80–90% of the width of the upper side of this growth ring was compression wood. Examination of cross sections taken 1.5–2.0 cm from the distal ends of the wood of original cuttings revealed a shift in the position of compression wood formation during the second season. As the distal portions of their stems were displaced from the vertical by curvature, cuttings of two clones produced compression wood on their lower sides in this region. Cuttings from the third clone, which showed the least basal curvature, continued forming compression wood on their upper sides in the distal portions of the original cuttings.