Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Müll. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.     

继代培养次数对杜梨生根能力和叶形态及其激素水平的影响

为了探索杜梨组培复幼变化规律,对10年生杜梨进行连续继代培养,统计不同继代次数杜梨丛生芽繁殖系数和生根率,观察记录叶片形态变化并测定内源激素含量。结果表明:(1)通过连续继代培养,杜梨丛生芽生根率由0提升到66.70%,繁殖系数由第1代的2.13提升到第10代的4.20。(2)叶片在继代第3次时出现裂刻且随后裂刻程度逐代加深;在继代过程中,丛生芽叶面积和叶脉数显著降低,叶周长和叶形指数呈先下降后上升的变化趋势。(3)丛生芽叶片内源IAA含量在继代第6次时达到46.39 ng·g-1,且显著高于初代丛生芽;随着继代次数的增加,叶片内源ZR呈先上升后下降的变化趋势,内源GA3含量没有发生显著性变化,而内源ABA含量逐渐降低;叶片IAA/ABA和IAA/ZR的值随着继代次数的增加而增加。(4)丛生芽叶片ABA含量和IAA/ZR与其生根率分别呈显著负相关和显著正相关关系,叶片裂刻数和IAA/ABA与生根率均呈现极显著正相关关系,而叶脉数与生根率则呈现极显著负相关关系。研究认为,连续继代培养可显著提高杜梨丛生芽的生根能力,并且与丛生芽叶形和激素含量及其比值有密切的关系,该研究结果为难生根植物无性繁殖以及树木复幼提供了重要技术借鉴。     

In vitro rooting of juvenile and mature<Emphasis Type="Italic"> Acacia mangium</Emphasis> microcuttings with reference to leaf morphology as a phase change marker

Rooting capacity of microshoots derived from a mature clone of Acacia mangium Willd. and from its juvenile progeny was compared in different in vitro conditions and in relation to leaf morphology as a phase change marker. Rooting capacity of the mature clone appeared to depend a lot on rooting medium composition, in contrast to the juvenile plant material. On a SH/3 derived medium with 4 µM IAA, the rooting rates varied significantly according to microshoot morphology and light regime starting from day 14. Maintaining the microshoots for 3 weeks in total darkness prior to transfer to the standard 16-h photoperiod increased the rooting rates overall. Mature-like microshoots of juvenile origin rooted in much lower proportions than the juvenile-like ones under the 16-h photoperiod, whereas no difference in rooting rates between the two morphological types was observed after 3 weeks in total darkness. The results were totally opposite for the mature clone for which 3 weeks in darkness, in contrast to the 16-h photoperiod, induced morphology-related differences in rooting rates with higher scores for the juvenile type. This study shows that under certain conditions, A. mangium microshoots from mature origin but with juvenile morphology can root in vitro with similar success rates (70%) to microshoots from juvenile plant material. However, the inconsistencies observed between the two age classes of microshoots in relation to photoperiod refute the use of foliar morphology as a reliable marker of rooting ability in A. mangium, although these two traits are classically referred to as phase change indicators.     

The influence of micrografting in vitro on tissue culture behavior and vegetative propagation of old European larch trees

The influence of a new micrografting method in vitro was tested by using long shoot meristems of adult 140 year old European larch trees grafted onto larch seedling rootstocks in sterile peat pellets. Five months after grafting and transfer of grafts to ex vitro conditions, new shoots from sprouting scions were re-established into tissue culture. The propagation behavior of shoots derived from these explants was compared with shoots established via bud culture from the same donor trees. Shoot explants from micrografts multiplied more rapidly than shoots explants from the original adult donor trees. Rooting experiments with cuttings from six micrografted clones resulted in 49.9 ± 11.9% rooting. Cuttings from donor trees invariably showed no root formation. The results confirm a rejuvenating influence of micrografting in larch.     

Effects of serial subculture in vitro on the endogenous levels of indole-3-acetic acid and abscisic acid and rootability in microcuttings of ‘Jonathan’ apple

Proliferating axillary shoots of the difficult-to-root apple cultivar Jonathan acquired an enhanced ability to form adventitious roots with increasing number of subcultures in vitro. The transition between the difficult-to-root and the easy-to-root condition occurred at the fourth subculture.Endogenous levels of free IAA and ABA in shoot tissues were analysed by gas chromatography/mass spectrometry/single ion monitoring (GC/MS/SIM) using negative ion chemical ionisation. Tissues from the mother plants grown in the glasshouse contained more IAA and ABA than those from tissue-culture material. After establishment in vitro there was no variation in the IAA content throughout the subcultures but a decrease in ABA content was observed after the fourth transfer. The IAA/ABA ratio increased from 0.2 in difficult-to-root shoots from the initial culture up to 0.7 in easy-to-root shoots from the long-term subculture.     

The lose of juvenility elicits adventitious rooting recalcitrance in apple rootstocks

For perennial woody plants, softwood cutting is an efficient technique for larger scale propagation and adventitious rooting of cuttings is one of the most crucial steps. To evaluate the significance of juvenility on adventitious rooting, rooting rates was compared between softwood cuttings collected from apomictic seedlings (juvenile), in vitro cultured plants (rejuvenated), suckers (juvenile like) and canopy shoots (adult) of reproductively mature trees in Malus xiaojinensis. After pre-treatment with indole-3-butytric acid (IBA) (3,000 mg L^?1) + H₂O₂ (50 mM), rooting rates in cutting from juvenile, juvenile like and rejuvenated donor plants were significantly higher (>90 %) than that from adult trees. The effects of IBA on adventitious rooting were enhanced significantly by exogenous H₂O₂. After 15 passages of in vitro subculture, the micro-shoots from adult phase explants were rejuvenated successfully, marked by the elevated expression of miR156 in the leaflets of the micro-shoots. But the rooting ability of rejuvenated micro-shoots was recovered delayed at the 18th or 21st passage of subculture. During the process of rejuvenation, the leaf indole-3-acetic acid contents and the expressions of rooting related genes CKI1, ARRO-1, ARF7 and ARF19 increased significantly. In contrary, the leaf abscisic acid contents decreased. A lack of juvenility is the most important limiting factor governing adventitious rooting of softwood cuttings in apple rootstocks.     

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

In this work, a combined HPLC-ELISA technique was used to associate in vitro rooting capacity of tree peony micro-cuttings with contents of cytokinin and auxin; the cytokinin mainly detected corresponded to the N⁶-benzyladenine which had been added to the multiplication medium. Rooting capacity of explants was favoured by a preliminary accumulation of endogenous IAA only when levels of the BA absorbed from the multiplication medium had decreased. Main shoots coming from a 5-weeks subculture fulfilled these hormonal conditions and were the best microcuttings for rooting (87% rooting). Main shoots coming from shorter cycles or axillary shoots coming from a 5-weeks cycle always contained high benzyladenine levels and had a low rooting capacity (25–55% rooting). Root induction was associated with an early peak of indole-3-acetic acid followed by a 10-fold lower peak of endogenous ribofuranosyl-isopentenyladenine. Only a low and transitory accumulation of isopentenyladenine occurred during root development, and this could explain the lack of shoot development. Root development was efficient, especially in a medium containing activated charcoal, which led to an almost 3-fold decrease of IAA contents in roots.Abbreviations AC activated charcoal - BA N⁶-benzyladenine - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - IBA indole-3-butyric acid - iP N⁶-(²-isopentenyl)adenine - RDM root development medium - RIM root induction medium - 9RIP 9--d-ribofuranosyl-iP - 9RZ 9--d ribofuranosyl-zeatin - Z zeatin     

Endogenous levels of abscisic acid and indole-3-acetic acid during in vitro rooting of Wild Cherry explants produced by micropropagation

Levels of endogenous ABA and IAA were quantified during the first week of in vitro rooting of Wild Cherry (Prunus avium L.) using IBA in the culture medium. Hormones were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme linked immunosorbent assay (ELISA), after a purification of the methanolic extracts by high-performance liquid chromatography (HPLC).Root primordia started to differentiate from day 5 at the basal part of the explants. ABA and IAA showed considerable changes and high levels were detected during the first week of culture. ABA levels increased transiently mainly in the apical part during root formation. Exogenous IBA was possibly transformed into IAA mainly in the basal part of the explants.     

Endogenous plant growth regulators and rooting capacity of different walnut tissues

Rooting ability of three different materials, namely cotyledonar linking areas, cotyledonary portions and embryonic microshoots from walnut seeds was determined. It was shown that while cotyledonar linking areas may be defined as a espontaneous rooting system, the other two tissue types correspond to inducible rooting systems, needing exogenous application of auxins for root induction. Endogenous zeatin and isopentenyl type cytokinins, indole-3-acetic acid (IAA), and abscisic acid (ABA) contents were determined in the three kinds of plant material prior to their culture. Our results show that the most active rooting systems has high levels of cytokinins, particularly dihydrozeatin (diH)Z and its riboside, and low levels of IAA and ABA. This situation could indicate that oxidative metabolism was taking place in cotyledonar linking areas, which is generally associated with the initiation phase of the rooting process. So that, we can assume that the induction phase is already overpassed in these tissues. On the contrary, the other two systems in which high levels of auxins and reduced levels of cytokinins were found, need to undergo the induction phase to be able to show root manifestation. In this case, the exogenous auxins are responsible for triggering this process, by enhancement of oxidative conditions. Therefore, it can be conclude that dynamics of growth regulators levels during the different phases of rooting are more important than specific single levels at fixed time.     

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L.

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.     

Shoot‐derived abscisic acid promotes root growth

The phytohormone abscisic acid (ABA) plays a major role in regulating root growth. Most work to date has investigated the influence of root‐sourced ABA on root growth during water stress. Here, we tested whether foliage‐derived ABA could be transported to the roots, and whether this foliage‐derived ABA had an influence on root growth under well‐watered conditions. Using both application studies of deuterium‐labelled ABA and reciprocal grafting between wild‐type and ABA‐biosynthetic mutant plants, we show that both ABA levels in the roots and root growth in representative angiosperms are controlled by ABA synthesized in the leaves rather than sourced from the roots. Foliage‐derived ABA was found to promote root growth relative to shoot growth but to inhibit the development of lateral roots. Increased root auxin (IAA) levels in plants with ABA‐deficient scions suggest that foliage‐derived ABA inhibits root growth through the root growth‐inhibitor IAA. These results highlight the physiological and morphological importance, beyond the control of stomata, of foliage‐derived ABA. The use of foliar ABA as a signal for root growth has important implications for regulating root to shoot growth under normal conditions and suggests that leaf rather than root hydration is the main signal for regulating plant responses to moisture.     

Metabolic changes during rooting in stem cuttings of Casuarina equisetifolia L.: effects of auxin, the sex and the type of cutting on rooting

Rooting in terminal shoot and lateral shoot cuttings from 10-year-old elite trees of Casuarina equisetifolia L. in different sex groups was achieved after 20 days when the basal ends of the cuttings were dipped for 3 h in 20 ppm indole-3-butyric acid (IBA). Shoots derived from male plants rooted better than their female and monoecious counterparts, and the lateral shoots were more responsive to rooting than the terminal shoots. During rooting, the metabolic activities varied in both lateral shoot and terminal shoot cuttings derived from plants under different sex groups. Peroxidase and polyphenoloxidase activities were high during root initiation and showed a sharp decline thereafter. The polyphenoloxidase activity was higher in the lateral shoot than the terminal shoot cuttings. The rooted plantlets survived and established well in the field.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting‐related growth regulators

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone‐free medium was conducted by high‐performance liquid chromatography. The endogenous concentration of auxin indole‐3‐acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root‐forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.     

In vitro shoot propagation of Dendrocalamus strictus nees

Multiple shoots were induced from seedling and axillary buds of mature plants of Dendrocalamus strictus on Murashige and Shoog's medium supplemented with BA and kinetin. About 35–45 shoots were obtained within 20–25 days from a nodal explant of seedling and 3–8 shoots were obtained from a nodal explant of mature plants in the primary culture. The seedling derived cultures were separated into groups of 5–7 and transferred to fresh subculture medium. Rooting of the shoots was achieved under in vitro and ex vitro conditions. 85–90% of rooting was achieved by the ex vitro method using IBA. This revised version was published online in June 2006 with corrections to the Cover Date.     

白花泡桐不定根发生过程中内源激素和RNA的变化

白花泡桐（Ｐａｕｌｏｗｎｉａｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．）成年型和幼年型茎切段体外培养时不定根发生过程中内源ＩＡＡ、ＣＴＫ和ＡＢＡ含量测定表明：幼年型材料中内源ＩＡＡ和ＣＴＫ的含量在诱导的第２天同时达到高峰,而成年型材料中ＩＡＡ和ＣＴＫ含量的高峰则在第４天出现．两种类型切段根原基出现的时间都与其内源ＩＡＡ和ＣＴＫ的高峰一致．幼年型材料的内源ＡＢＡ含量在第４天达到高峰,随后迅速下降．成年型材料中内源ＡＢＡ则逐步下降．成年型和幼年型材料中ＲＮＡ的变化相同,在诱导的第２天稍有下降,随后显著增加．结果显示,不定根的发生与其内源激素和ＲＮＡ的变化密切相关．     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

Maturation and rejuvenation of Coriandrum sativum shoot clones during micropropagation

Three clones of Coriandrum sativum L. shoots were obtained from three seedlings and micropropagated alternately on modified MS media containing kinetin only and kinetin plus indolyl-3-acetic acid (IAA). During the first 9 months of culture the shoots possessed the juvenile phenotype after which a sharp transition to mature phenotype occurred. In 15–17 months this was followed by shoot necrosis and decrease in number of shoots in the clones, leading to death of the clones.Conditions of in vitro culture tripled the length of the juvenile period. Mature phase of the shoots was stable in that no reversion to the juvenile phase was observed. Partial rejuvenation of mature shoots took place owing to formation of adventitious shoots in the callus formed at the shoot base. However maturation of such rejuvenated adventitious shoots took place much more rapidly in comparison with micropropagated juvenile shoots derived from seedlings. Reduction of the morphogenic potential of the mature shoots after 15–17 months of subculturing, an increase in number of abnormal shoots and shoot necrosis indicated physiological ageing of the clones.Data presented in the paper provide evidence of the clone ageing phenomenon during prolonged subculture in vitro.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation and rooting of <Emphasis Type="Italic">Acacia mangium</Emphasis> microshoots from juvenile and mature origins

Acacia mangium microshoots from juvenile and mature genotypes were micropropagated through a regular subculture regime for more than 3 yr in vitro. Average multiplication rates of 5.5 for the juvenile source and of 3.9 for the mature clone were obtained during this period on the 6-benzylaminopurine-enriched multiplication medium. Although the juvenile material displayed higher potential for axillary shoot and root formation than the mature clone overall, the differences were not statistically significant with noticeable variations in the course of time from one subculture to another. On specific rooting media, the juvenile material rooted overall in greater proportions than the mature material, notwithstanding noteworthy interactions between the age of the plant material and the various experimental factors tested, i.e. sucrose concentration, macrosalt formulation and light regime. The stimulating effect of darkness on juvenile plant material rooting rates was more obvious than for the mature clone, which responded more inconsistently. Addition of 4 μM indole-3-acetic acid, indole-3-butyrie acid, or 1-naphthaleneacetic acid in the rooting medium significantly increased the proportion of rooted microshoots of both origins. The rooting criteria observed were also prone to vary depending on the experimental date. The data indicate that rooting of juvenile and mature Acacia mangium materials have average rates of 90% and 77%, respectively. These are high enough to consides possible applications of these procedures toward operational activities.