Binucleate Rhizoctonia (Ceratorhiza spp.) induce adventitious root formation in hypocotyl cuttings of Pinus sylvestris L.

Adventitious rooting of Scots pine (Pinus sylvestris L.) hypocotyl cuttings was promoted by binucleate Rhizoctonia (BnR) and to a lesser extent by ectomycorrhizal fungi. Four BnR isolates (251, 266, 268 and 269) differentiated root meristems and significantly induced adventitious rooting in young derooted seedlings. Rooting rates were significantly higher in BnR treatments than in either indol-3-butyric acid (IBA) pretreatment (200 µm) or co-cultivation with the ectomycorrhizal fungi Suillus bovinus or Laccaria bicolor. In pre-IBA-treated cuttings, adventitious root numbers were higher in the treatments with isolates 251 and particularly 268, while more similar in other BnR treatments. In the 251, roots emerged from distal positions along the hypocotyl and not from the cut base as in the other three BnR isolate treatments. Loss of turgor and hypocotyl wilt was commonly detected up to the point of root initiation. Lateral roots and dichotomous short roots subsequently appeared at high frequency from primary adventitious roots. Mechanisms involved in root meristem differentiation, e.g. auxin production, wound response and oligosaccharide signals, are discussed with respect to host-fungal signalling mechanisms. These beneficial BnR and ectomycorrhizal fungi could provide promising new tools in the development of efficient clonal propagation methodology for this highly recalcitrant Pinus species.     

Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species

We characterized SCARECROW-LIKE genes induced by auxin in rooting-competent cuttings of two distantly related forest species (Pinus radiata D. Don and Castanea sativa Mill.) before the activation of cell division that results in adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family and show a strong similarity to the SCARECROW-LIKE proteins, indicating conserved functions of these proteins. Quantitative RT-PCR analysis showed that these genes are expressed at relatively high levels in roots. Induction of increased mRNA levels in rooting-competent cuttings of both species in response to exogenous auxin was observed within the first 24 h of the root induction process, a time when cell reorganization takes place, but before the resumption of cell division and the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes play a role during the earliest stages of adventitious root formation.     

Distinct effects of auxin and light on adventitious root development in Eucalyptus saligna and Eucalyptus globulus

Adventitious rooting is essential for vegetative propagation of woody species. We studied the effects of auxin and light on the development of adventitious roots in cuttings obtained from seedlings of Eucalyptus saligna Smith and E. globulus Labill in an attempt to characterize the adventitious rooting process and identify factors controlling rhizogenesis. Root development was scored as rooting percentage, root density (roots per rooted cutting), mean rooting time and root length. In both species, rooting time was reduced in the presence of auxin. Cuttings from 2-month-old E. saligna seedlings were responsive to lower auxin concentrations than comparable cuttings from E. globulus seedlings. Cuttings from 3-month-old E. saligna seedlings rooted promptly and rooting was not significantly affected by light conditions. In contrast, rooting of cuttings from 3-month-old E. globulus seedlings exhibited recalcitrant behavior and no roots were formed if illuminated during the root formation phase. Effective root regeneration of E. globulus cuttings was obtained by a 4-day exposure to 10 mg l(-1) IBA and culture in darkness during the root formation step. Loss of rooting capacity with seedling age was more pronounced in E. globulus than in E. saligna. The possibility of switching adventitious rooting off and on by manipulating light regime and exogenous auxin supply in E. globulus, and the constitutive nature of rooting in E. saligna may provide useful models for examining the rooting process at the biochemical and molecular levels in Eucalyptus.     

Effects of exogenous diamines on the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine in vitro

Production of free and conjugated polyamines by two ectomycorrhizal fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr., was studied in vitro. Spermidine was the main polyamine in the mycelium of both fungi. Paxillus involutus also produced large amounts of the diamine putrescine, whereas Pisolithus tinctorius contained traces of the diamine cadaverine and released into the culture medium an unknown compound probably related to cadaverine or N-methylputrescine. Both fungi accelerated adventitious root formation and increased subsequent root growth of Scots pine (Pinus sylvestris L.) hypocotyl cuttings in vitro. Exogenous cadaverine enhanced rooting caused by Pisolithus tinctorius and also promoted mycorrhiza formation by the fungus. Putrescine and Paxillus involutus had a synergistic effect on root initiation, but not on subsequent root growth. We conclude that specific diamines may be involved in the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine, and that the effects of specific exogenous polyamines are dependent on the fungal strain and its ability to produce these compounds. The finding that Paxillus involutus enhanced rooting and root growth without mycorrhiza formation indicates that fungal-induced rooting is not necessarily related to visible mycorrhiza formation.     

Ectomycorrhizal fungi and exogenous auxins influence root and mycorrhiza formation of Scots pine hypocotyl cuttings in vitro

We studied the ability of the ectomycorrhizal (ECM) fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr. (Strain H), to produce indole-3-acetic acid (IAA) and to affect the formation and growth of roots on Scots pine (Pinus sylvestris L.) hypocotyl cuttings in vitro. Effects of indole-3-butyric acid (IBA) and the auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), on rooting and the cutting-fungus interaction were also studied. Both fungi produced IAA in the absence of exogenous tryptophan, but the mycelium and culture filtrate of Pisolithus tinctorius contained higher concentrations of free and conjugated IAA than the mycelium and culture filtrate of Paxillus involutus. Inoculation with either fungus or short-term application of culture filtrate of either fungus to the base of hypocotyl cuttings enhanced root formation. Inoculation with either fungus was even more effective in enhancing root formation than treatment of the hypocotyl bases with IBA. Fungal IAA production was not directly correlated with root formation, because rooting was enhanced more by Paxillus involutus than by Pisolithus tinctorius. This suggests that, in addition to IAA, other fungal components play an important role in root formation. Treatment with 5 microM TIBA increased the rooting percentage of non-inoculated cuttings, as well as of cuttings inoculated with Pisolithus tinctorius, perhaps as a result of accumulation of IAA at the cutting base. However, the marked reduction in growth of Pisolithus tinctorius in the presence of TIBA suggests that the effects of TIBA on rooting are complicated and not solely related to IAA metabolism. The high IAA-producer, Pisolithus tinctorius, formed mycorrhizas, and the IBA treatment increased mycorrhizal frequency in this species, whereas TIBA decreased it. Paxillus involutus did not form mycorrhizas, indicating that a low concentration of IAA together with other fungal components were sufficient to stimulate formation and growth of the roots, but not the formation of ECM symbiosis.     

Genetic variation in rooting ability of loblolly pine cuttings: effects of auxin and family on rooting by hypocotyl cuttings

After about 20 days, hypocotyl cuttings from 20-day-old loblolly pine (Pinus taeda L.) seedlings rooted easily in the presence of the auxin indole-3-butyric acid (IBA), with roots forming directly from xylem parenchyma. In contrast, woody cuttings from 1-2-year-old hedged seedlings formed roots indirectly from callus tissue in 60-90 days, but IBA had little effect on rooting. Variation in rooting among hypocotyls from both half- and full-sib families was highly significant in response to IBA, and rooting did not occur within 20 days unless IBA was applied. Hypocotyls from poor rooting families tended to produce fewer roots per cutting than hypocotyls from good rooting families. Rooting by woody cuttings and hypocotyl cuttings from the same nine full-sib families was weakly correlated, raising the possibility that at least some common genetically controlled processes were affecting rooting by both types of cutting. The phytotropin N-1-naphthylphthalamic acid (NPA), supplied at 1 micro M with 10 micro M IBA, significantly inhibited rooting by hypocotyl cuttings from both good and poor rooting families, but there was no significant family x treatment interaction. Family variation in rooting ability may be a function of the frequency of occurrence of auxin-responsive cells in the hypocotyls.     

兴安圆柏嫩枝插穗生根相关内源激素特征分析

为了提高兴安圆柏扦插生根率,探讨兴安圆柏的扦插生根机理,以改进的HPLC法测定了兴安圆柏嫩枝插穗生根过程中4种内源激素的含量变化。结果表明,外源激素处理促进了插穗内IAA合成,显著提高了插穗内IAA含量、IAA/ABA值,ABA含量变化不明显;低浓度的ZT促进生根,插穗生根过程中ZT含量较低,NAA处理使ZT含量比对照显著降低;外施激素降低了生根初期插穗内GA含量,促进了愈伤组织和根原基形成,提高了不定根形成期插穗内GA含量,有利于不定根生长。     

Clonal propagation of <Emphasis Type="Italic">Dalbergia sissoo</Emphasis> Roxb. and associated metabolic changes during adventitious root primordium development

A study was conducted on the twelve clones of shisham (Dalbergia sissoo Roxb.). These clones were obtained from India and Nepal. Single-node leafy cuttings were prepared from the vegetative multiplication garden to examine the clonal variation, effect of IBA treatment on rooting response and associated metabolic changes during adventitious root formation. A remarkable and significant variation was observed due to treatment of 2,000 ppm IBA in the rooting parameters. Clonal variations were also significant for root and shoot growth while length of root was insignificant. Among the twelve clones studied; C3 (Tulsipur, Gonda, Uttar Pradesh, India) and C4 (Laxmipur, Gonda, Uttar Pradesh, India) clone cuttings have given the highest rooting response. Interaction (clone × IBA) was significant only for production of number of roots per cutting. Periodic sampling for clone C3 was performed at 0, 7, 14, 21, 28 and 35 days to examine the contents of total soluble sugars, starch, protein and peroxidase (PER) activity in the rooting zone of cuttings (∼0.5 cm) during adventitious root primordium development. A significant increase in all the metabolic activities was noted due to IBA. Total soluble sugars and starch contents of cuttings decreased with the passage of time. Protein content and PER-activity started to increase in the early stage and reached the highest level on day 21, followed by a decline at the 35th day of sampling. These trends were common for both IBA treated and untreated cuttings. Protein content and PER-activity remained higher in the rooting zone of IBA treated cuttings. Overall these findings suggested that exogenous application of IBA may have activated carbohydrate metabolism for release of energy, while protein and PER-activity were necessary for cell division and differentiation during adventitious root primordium initiation and development in the rooting zone of cuttings.     

三种杨树扦插生根期间内源激素水平的比较研究

用高效液相色谱（ＨＰＬＣ）和气相色谱（ＧＣ）法，分析银白杨、毛白根和山杨的对照、ＮＡＡ处理和ＮＡＡ＋ＢＡ处理硬枝插穗（芽、皮）的内源ＡＢＡ、ＩＡＡ和玉米素含量在扦插生根期间的变化。试验表明，硬枝扦插生根是潜伏根原基存在与否和皮部内源激素平衡两方面的原因决定的。银白杨皮部的ＩＡＡ／ＡＢＡ比值和玉米素含量高，潜伏根原基有迅速恢复生长的条件，能自发生根成活。毛白杨的ＩＡＡ／ＡＢＡ比值和玉米素含量都低，需     

杂种落叶松连续繁殖与插穗生根关系的生理研究

观察了日×长杂种落叶松连续繁殖插穗不定根发育进程,研究了连续繁殖对插穗生根力衰退的阻滞效应,并分析了一轮采穗圃与原株采穗圃插穗不定根发育期内源激素含量的动态变化。结果表明:扦插后13 31 d是愈伤组织形成和不定根原始体分化发育的关键期,此时一轮分生株愈伤组织形成和不定根发育均优于原株。连续繁殖对插穗生根性状有显著作用,尤其是生根率很低的原株,经过一轮繁殖后,生根性状有极显著提高。连续繁殖影响插穗自身激素含量,尤其是IAA,经过一轮繁殖后,含量明显高于原株。从不定根发育过程中激素的动态变化看,一轮分生株插穗(IAA+GA3+ZR)/ABA比值在不定根发育前期明显高于原株,与生根率的变化一致,可用来衡量不同繁殖次数插穗生根性状的优劣。     

Auxin and ethylene regulation of diameter growth in trees

Recent studies on the phytohormonal regulation of seasonal cell-division activity in the cambium, primary-wall radial expansion of cambial derivatives, differentiation of xylem cells, and growth of the cortex in forest trees of the north temperate zone are reviewed. Indol-3-ylacetic acid (IAA, auxin) has been characterized by combined gas chromatography-mass spectrometry (GC-MS) in the cambial region of Abies balsamea, Pinus densiflora, Pinus sylvestris and Quercus robur. All of the evidence supports the hypothesis that developing leaves and extending shoots are primary sources of IAA. The rate of ethylene emanation varies among conifer species when adjoining phloem and cambial tissues are incubated in vitro. The cambium from young cuttings of Abies balsamea produces more ethylene than that from older cuttings. Ethylene production by seven-year-old Abies balsamea cambium is substantially increased in vitro when the tissue is provided with exogenous 1-aminocyclopropane-1-carboxylic acid and IAA. In response to elevated ethylene concentrations, cortex growth is accelerated in both hardwood and conifer seedlings. Ethrel (2-chloroethylphosphonic acid) increases ray size and ray-cell number and promotes traumatic resin-canal development in xylem. In Ulmus americana, endogenous ethylene concentrations are inversely correlated with cambial activity. Ethylene decreases vessel diameter in Acer negundo, Acer platanoides and Ulmus americana. Several studies suggest that ethylene has a role in regulating reaction-wood formation in both conifers and hardwoods.     

Metabolic changes during adventitious root primordium development in <Emphasis Type="Italic">Tectona grandis</Emphasis> Linn. f. (teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment

Aging of the donor tree decreased adventitious root formation in shoot cuttings of Tectona grandis Linn. f. (teak). Exogenous application of auxins, i.e., α-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) has a significant positive effect on the percentage of rooting. The maximum percent rooting was obtained with 4,000 ppm IBA as compared to other treatment. Significant increase in root number was recorded in shoot cuttings treated with 4,000 ppm NAA. The overall rooting response was better in the treatment with IBA rather than with NAA. Further periodic samples (0, 10, 20, and 30 days) were taken to assess the total soluble sugar, starch, protein, and peroxidase (PER) activity in the rooting zone of shoot cuttings of teak during adventitious root formation. Application NAA and IBA to shoot cuttings resulted in an increase in the level of total soluble sugar, starch, protein, and PER-activity in the rooting zone. The stored carbohydrates were utilized during adventitious root formation. Hence, total soluble sugar and starch contents of cuttings, irrespective of age of donor plants, decreased with the passage of time in cuttings planted for rooting. Significant fluctuations were observed in the protein content of cuttings during the time of root induction. There was an increase in the protein content with the passage of time from the day of planting up to its 20th day, followed by a sharp decline in the protein content of cuttings at the 30th day of planting, irrespective of the age of donor plants or the treatment of cuttings with auxins. Irrespective of donor plant age, PER-activity in the cuttings increased from the day of their planting for rooting up to the 20th day, and then declined at its 30th day of planting. It was interesting to note that PER-activity remained higher at all stages in the cuttings of 2-month-old seedlings which rooted profusely as compared to the cuttings of 15- and 30-year-old donor plants those rooted poorly. This study suggested that the exogenously applied NAA and IBA at different concentration seems to activate sugar metabolism for release of energy, protein and PER-activity which are necessary for cellular division and differentiation during adventitious root primordium initiation or development in the rooting zone of shoot cuttings.     

Variation in Shoot Anatomy and Rooting Behaviour of Stem Cuttings in Relation to Age of Donor Plants in Teak (Tectona grandis Linn. f.)

Shoot anatomical features viz., number of vessel elements, width and length of vessel and fibre elements were studied in relation to juvenility/maturity of teak (Tectona grandis Linn. f.). The findings show significant differences in these features, which indicates that variation in shoot anatomy can be used as a reliable indicator of juvenility/maturity in teak. The effect of age of donor plants and application of auxins on adventitious root formation in single noded leafy stem cuttings of teak was also investigated. Aging of donor trees suppressed rooting and sprouting of cuttings, but increased callus formation at the base of cuttings. Per cent rooting and per cent sprouting in cuttings decreased as age of donor plants increased from 2-months to 15-years and up to 30-years. IBA treatment promoted per cent rooting as well as per cent sprouting, NAA promoted only rooting and it suppressed sprouting. The effect of auxin treatment on rooting and sprouting of teak stem cuttings varied with the age of donor plants. Auxin and its concentration, which suppressed rooting and sprouting in cuttings of younger donor plants, actually promoted rooting when cuttings were taken from more mature donor plants. It appears that the auxin requirement for causing and promoting rooting in teak cuttings increased with increasing age of donor plants. The higher auxin requirement for causing and promoting rooting in cuttings of older trees may be due to a decrease in the content on endogenous auxins or decreased sensitivity of aging tissues to rooting promoters and /or accumulation of inhibitory substances which inhibit rooting.     

细柄蕈树嫩枝扦插生根过程中内源激素动态特性研究

以3年生细柄蕈树实生苗嫩枝为试验材料,运用酶联免疫吸附测定法(ELISA),研究其在扦插生根过程中内源激素含量的变化,旨在揭示细柄蕈树扦插生根的机理。结果表明,与CK相比,以质量浓度300 mg/L的NAA处理插穗,能有效缩短其生根周期;扦插生根过程中,NAA处理与CK的几种内源激素(IAA、ZT和ABA)含量随时间变化的趋势相同,均呈正"V"形变化;NAA处理可调节内源激素,促进不定根的形成和发育;不定根形成期间,NAA处理与CK的IAA/ZT、IAA/GA3和IAA/ABA谷值的出现时间(20 d和30 d)与愈伤组织形成期重合,内源激素的这种节奏性变化反映了生根与生理之间的协同性。     

Effect of position,size of cuttings and environmental factors on adventitious rooting in neem (Azadirachta indica A. Juss)

The effect of adventitious rooting of proximal and distal position of cuttings of neem of different length and diameter (with 1000 ppm IBA treatment) was studied. Cuttings from the distal end rooted better than the proximal end, probably due to higher endogenous auxin level. Cuttings of 25 cm length and 0.5 cm diameter from the distal end gave 100% rooting. The percentage of rooting decreased significantly when the length of the cutting was reduced, and no rooting was observed in 5 cm long cuttings irrespective of the position, suggesting a sub-optimal level of endogenous auxin and other root-inducing factors in small cuttings. Low radiation may have had a significant role in the induction of rooting.     

Rice Bran Extract as a Synergist of Adventitious Rhizogenesis in Teak

Abstract

Graded doses of IBA (200, 400, and 600 ppm), rice bran extract (0.075%, 0.150%, 0.225% and 0.300%, w/v) and their all possible interactions significantly influenced adventitious rhizogenesis in semi-hardwood shoot cuttings of Tectona grandis. Treatments with 600 ppm IBA and 0.150% rice bran extract proved optimum and increased adventitious rooting by 442% and 49%, respectively, over the control. The regression analysis for various characteristics of propagules indicated a direct role of constituent(s) of rice bran in the process of adventitious rhizogenesis, explaining the total observed variations to the extent of 82.34% in sprout length (p < 0.05), 94.26% in rooting percent (p < 0.05), 88.38% in root number (p < 0.1) and 75.25% in root length (p < 0.1) to be a function of exogenous graded doses of rice bran extract. A very strong synergism between IBA and rice bran was also observed, for a combined application of 600 ppm IBA and 0.150% rice bran extract enhanced adventitious rooting by 1735% over the control. Thus, rice bran extract not only emulates auxin but also acts as rooting synergists.     

Early and late root formation in epicotyl cuttings of Pinus sylvestris after auxin treatment

Auxin stimulated rooting of epicotyl cuttings of Pinus sylvestris irrespective of the physiological stage of the plant from which the cuttings were taken. The epicotyl cuttings were divided into two groups, those that differentiated roots early (within 6 weeks after cutting) and those that differentiated roots late (more than 6 weeks after cutting). Auxin treatment significantly stimulated the frequency of early rooting of epicotyl cuttings, whereas it had no effect on the frequency of late rooting of epicotyl cuttings. The number of roots per rooted cutting was significantly higher after auxin treatment both on early and late rooted cuttings. Anatomical studies showed that most of the roots on both early and late rooted cuttings developed from wound tissue. However some types of rooting were found only after auxin treatment of early rooted cuttings, e.g., roots that developed from resin duct wound tissue or from vascular tissue or in the pith.     

无患子硬枝扦插进程插穗内源激素和多酚类物质含量变化

为了解无患子硬枝扦插生根机制,以1年生无患子硬枝作为扦插材料,在扦插第0—80天调查愈伤组织发育和根系形成情况,测定插穗的腋芽(嫩叶)以及基部2cm长度的韧皮部内源激素和多酚类物质的含量。结果表明,无患子硬枝扦插第10天开始出现愈伤组织,第50天愈伤率达到最高值83.33%;第40天开始出现生根插穗,生根率为6.67%,随后不定根数量迅速增加,第70天生根率达到86.67%,此时平均有5.33条。之后生根数量不变,根系仍然在生长,且扦插后根系效果指数持续增高。插穗韧皮部内源激素含量变化较复杂。整体来看,高含量赤霉素(GA3)抑制愈伤组织和不定根形成,且在根原基发生期和不定根形成关键期达到峰值,在不定根速生期持续下降;高含量生长素(IAA)促进不定根的形成,且在扦插后第20天,即根原基发生期,达到峰值(92.7μg/g);玉米素核苷(ZR)对无患子硬枝扦插过程的生理作用较复杂,低含量的ZR有利于根原基的发生和不定根速生,但高含量的ZR促进不定根形成。韧皮部ZR/IAA在扦插后第20—40天呈下降趋势,第40—60天快速上升,促进不定根生长;GA3/IAA整体呈现抛物线形下降趋势,特别是在根原基发生期和不定根速生期,下降速率更快,以促进不定根的生长。无患子硬枝插穗生根进程中,多数多酚类物质对愈伤组织形成、根原基发生及不定根形成有极显著抑制作用,没食子酸抑制效果稍弱;插穗内多种激素和多酚类物质的含量均发生了变化,且对插穗生根产生重大影响。总之,插穗根原基发生和不定根形成等关键时期,IAA含量升高,GA3、ZR以及多酚类物质含量降低,植物体内多种内源物质此消彼长,达到动态平衡,共同促进插穗生根。     

Effects of IBA on the rooting of branch cuttings of Chinese jujube (<Emphasis Type="Italic">Zizyphus jujuba</Emphasis> Mill.) and changes to nutrients and endogenous hormones

‘Zhongqiusucui’ jujube secondary shoots were treated with 3-indolebutyric acid (IBA) at three concentrations, 500, 1000 and 1500 mg/L. Results show that IBA could significantly enhance rooting and root characteristics of cuttings and were best with IBA at 1500 mg/L. In the rooting process, the formation of adventitious roots was related to the consumption and accumulation of nutrients (soluble sugars and proteins) and the changes in endogenous hormones in phloem, leaf tips and leaf bases. The rooting of cuttings had a positive correlation with the consumption of soluble sugars during the period of callus formation and with the accumulation of soluble sugars during adventitious root formation and growth. Rooting was positively related to the breakdown of soluble proteins in the phloem when the callus formed, and had a positive correlation with its accumulation during adventitious root formation and growth. Leaf tips and leaf bases showed a reverse trend in changes of soluble protein. However, together with the phloem, leaf tips and leaf bases regulated and controlled the formation and development of adventitious roots. The main activities of soluble proteins exist in the leaf tips as this was the main source of soluble proteins. The relation between rooting and IAA (indole-3-acetic acid) content in phloem was positive and thus a high concentration of IAA could benefit the induction and formation of adventitious roots. However, rooting was negatively related with ABA (abscisic acid) and GA (gibberellic acid) and a high concentration of both could inhibit the induction and formation of adventitious roots. Rooting had a positive correlation with phloem IAA/ABA ratios, and higher ratios could improve rooting. Low concentrations of ZR (zeatin riboside) triggered the induction of adventitious roots, while higher concentrations promoted root growth. Endogenous hormones in leaf tips and bases had an impact on rooting. The activities of endogenous hormones mainly existed in leaf tips because they play a major role in the production and consumption of IAA and its ABA content increased during rooting. The ZR in leaf tips influenced the rooting of cuttings, especially in the callus formation and rooting stage. Leaf tips were the main source of GA.