Influence of auxins and darkness on in vitro rooting of micropropagated shoots from mature and juvenile Acacia mangium

The influence of total darkness versus a 16/8 photoperiod and of auxins added to the culture medium on the in vitro root formation capacity of Acacia mangium microshoots of juvenile and mature origin was examined. Rooting of the mature clone was significantly increased by exposing the microshoots to auxins (4 and 6 μM IAA or IBA) in darkness, while the promoting effect of darkness combined with 4 μM IAA was more time-restricted for the juvenile-origin microshoots. Overall, the latter rooted in greater proportions than those from the mature source. Maintaining the microshoots of both origins on auxin supplemented medium in darkness resulted in a greater number of adventitious roots formed than under the standard 16/8 lighting conditions. On the other hand, light stimulated root elongation. These results are discussed mainly from the viewpoint of auxin metabolism in relation to adventitious root formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

<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.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of two <Emphasis Type="Italic">Eucalyptus urophylla X Eucalyptus grandis</Emphasis> mature clones

Abstract   The rooting capacity of microshoots derived from two mature Eucalyptus urophylla X Eucalyptus grandis half-sib clones kept for 3 y under intensive micropropagation was assessed in different in vitro conditions. A first set of experiments established that clone 147 microshoots rooted earlier and in greater proportions, while producing more adventitious roots overall than their homologs from clone 149. Modifying the composition of the basal 1/2-MS-derived rooting medium by 1/4-MS or Knop macronutrients, or reducing sucrose concentration to 10 g l⁻¹ did not enhance the rooting rates. However, together with the growth regulators added, they had a significant effect on the number of adventitious roots formed. With rooting rates reaching 81%, the higher rootability of clone 147 over clone 149 was further confirmed by the second set of experiments with significant effects of the various auxins tested and strong clone × auxin interactions on the proportions of rooted microshoots and on the number of adventitious roots. The best rooting scores were given by 5 μM indole-3-butyric acid (IBA) and 12.5 μM 1-naphthaleneacetic acid (NAA), whereas the microshoots exposed to 5 or 12.5 μM indole-3-acetic acid (IAA) were less responsive. Lower light intensities did not improve significantly root capacities, although differences might exist according to the genotype. Overall, root and shoot elongation was stimulated by light. At the end of the experiment, the rooted microshoots were markedly taller than the non-rooted ones, with significant influences of auxins and light intensity, and to a lesser extent, of the genotypes.     

Adventitious root formation in woody plant tissue: The influence of light and indole-3-butyric acid (IBA) on adventitious root induction in <Emphasis Type="Italic">Betula Pendula</Emphasis>

Summary The effects of auxin concentration and photoperiod on rooting were examined with a view to establishing a rooting regime for Betula pendula shoots cultured in vitro. Optimum concentrations of indole-3-butyric acid (IBA) were determined: the effects of a 16-h photoperiod and a pretreatment of 8d total darkness were examined. Maximum rooting rates and rooting densities (root number) were achieved using relatively low levels of IBA (0.39–0.74 μM). Both the dark and the light regimes produced roots, higher yields occurring with the latter. Maximum rooting percentage was reached after 30 d growth. in the light-treated cultures.     

Effect of Auxins on in vitro Rooting of Plumbago Zeylanica: Peroxidase Activity as a Marker for Root Induction

Induction of rooting in the microshoots of Plumbago zeylanica was achieved on halfstrength basal Murashige and Skoog's medium supplemented with 0.25 mg dm^–3 indole-3-butyric acid. Rooting was totally inhibited when the microshoots were cultured in vitro under continuous light, however, maximum percentage of microshoots rooted when incubated in continuous light for 4 weeks before transfer to the rooting media. Peroxidase activity increased markedly during root induction indicating a key role of peroxidase in rooting of microshoots of Plumbago zeylanica in vitro.     

In vitro rooting of Psoralea corylifolia Linn: Peroxidase activity as a marker

Induction of rooting in microshoots of Psoraleacorylifolia was achieved within 6–8 days of cultureon half-strength basal Murashige and Skoog's(1962) medium supplemented with 0.005–0.01 mg/lindole-3-acetic acid (IAA) and 2% (w/v) sucrose. Rooting was drastically reduced and friable callusformed at the cut end of the microshoots when themedium was supplemented with a higher concentration ofauxin. Rooting was totally inhibited when themicroshoots were cultured in vitro undercontinuous light. However, the maximum percentage ofmicroshoots rooted when incubated in continuous lightfor 4 weeks before transfer to the rooting media.Peroxidase activity increased considerably duringroot induction indicating a key role of peroxidase inrooting of microshoots of Psoralea corylifolia invitro.     

In vitro seed culture and seedling development of Calopogon tuberosus

A major obstacle to native orchid production is difficulty in seed germination. Culture media and light effects on seed germination of Calopogon tuberosus var. tuberosus, a native orchid with horticultural potential, were studied. Culture media included Knudson C, Malmgren modified terrestrial orchid, and PhytoTechnology orchid seed sowing. Effects of 8 weeks continual darkness, 8 weeks 16-h photoperiod, 2 weeks dark followed by 6 weeks 16-h photoperiod, 4 weeks dark followed by 4 weeks 16-h photoperiod, and 6 weeks dark followed by 2 weeks 16-h photoperiod were examined. Percent seed germination was highest on Knudson C after 8 weeks culture; however, seedling development was enhanced on PhytoTechnology seed sowing medium during 8 weeks culture under a 16-h photoperiod. This suggests that while KC and darkness promoted seed germination, P723 and light enhanced further seedling development. Seedlings of C. tuberosus readily acclimated to greenhouse conditions.     

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACT

Pinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.     

Phenolic Content of Microcuttings of Adult Chestnut along Rooting Induction

From the same adult 80-year-old tree of chestnut (Castanea sativa Mill.) 2 types of material have been taken and micropropagated. This resulted in in vitro easy-to-root microshoots (basal shoot origin - BS), and hard-to-root microshoots (crown shoot origin - CR). In these shoots, the phenolic contents were analysed at 0, 2, 5 and 8 days after in vitro rooting induction by 2 minute-dipping into an indole-3-butyric acid (IBA) solution (4.9 mM) and subsequent culture in a hormone-free rooting medium. The variation of the phenolic content along the adventitious rooting process differs between CR and BS microshoots for tannin, flavonol and elagic acid concentrations, which could be related to their differential rooting capacity.     

Micrografting of <Emphasis Type="Italic">Protea cynaroides</Emphasis>

The inability to induce rooting of in vitro-established Protea cynaroides microshoots has prevented the production of complete plantlets. A successful shoot-tip micrografting technique was developed using in vitro-germinated P. cynaroides seedlings as rootstocks and axenic microshoots established from pot plants as microscions. Thirty-day old seedlings, germinated on growth-regulator-free, half-strength Murashige and Skoog medium, were decapitated and a vertical incision made from the top end. The bottom ends of microshoots established on modified Murashige and Skoog medium were cut into a wedge (‘V’) shape, and placed into the incision. The micrografted explants were cultured in a growth chamber with the temperature adjusted to 25 ± 2°C, with a 12-h photoperiod. Best results were obtained by placing the microscions directly onto the rootstock without any pre-treatments. Dipping the explants in anti-oxidant solution or placing a layer of medium around the graft area led to the blackening of the microscion.     

In vitro reinvigoration of mature olive trees (Olea europaea L.) through micrografting

Summary Portions (1.0–1.5 cm long) of terminal shoots from selected mature treesOlea europaea L. cv. Arbequina, micrografted in one phase ontoin vitro juvenile shoots, resulted in the restoration of shoot-bud proliferation and rooting competence. Although higherin vitro survival rates were obtained after a second repeated micrografting, the reinvigoration ratio of the regenerated shoots, indicated by proliferation and rooting ability, was not improved after two phases of micrografting. Thus, one-phase micrograft allows for a successful micropropagation system for olive trees. The cuttings obtained from successive pruning of plants produced through micrografting and growth in soil showed complete restoration of rooting competence, with rooting percentages similar to those of juvenile microshoots.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Acer grandidentatum</Emphasis> Nutt.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.     

Onset of in vitro rhizogenesis response and peroxidase activity in Zingiber officinale (Zingiberaceae)

The induction of rooting in microshoots of Zingiber officinale cvs. Suprava, Turia local, Suruchi and V3S18 was achieved on half-strength basal Murashige and Skoog's medium supplemented with 0.5-1.0 mg/l either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) and 2% (w/v) sucrose within 7-9 days of culture. Rooting was inhibited when the microshoots were cultured under higher concentration of auxins. The microshoots cultured on medium supplemented with NAA induced large number of thin root hairs with friable calluses within 6-7 days. Peroxidase activity was determined during root induction (0-day to the 10th day at every 2 day interval) from microshoots derived in vitro. The activity was minimum in the inductive phase (primary) and at the maximum level during the root initiative phase. These finding may be useful in monitoring the rooting behaviour in microshoots derived from different subculture and peroxidase activity as a marker for root initiation.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Rooting of Sitka spruce cuttings from hedges,and after chilling

Summary Four clones of Sitka spruce (Picea sitchensis (Bong.) Carr.) were established from cuttings of two and four-year old material in 1968. Within each clone half the trees were randomly hedged at 1 m in 1977. Cuttings from hedges rooted more freely than cuttings from the lower crown which, in turn, rooted more readily than upper crown cuttings. Rooting occurred most readily during January and early February. Concentrations of sugars in stems and foliage showed little correlation with rooting.Chilling must be completed for most rapid rooting of dormant Sitka spruce cuttings and this requirement can be satisfied by 10 weeks at 2°C.     

Micropropagation of juvenile and adult Annona squamosa

A micropropagation system for Annona squamosa L. (Sugar Apple) using hypocotyls of seedlings and nodal cuttings from 3-year-old plants was developed. Shoot proliferation was achieved with Woody Plant Medium supplemented with BA. Silver thiosulphate was added at 0.5 mg l^–1 to control leaf abscission. Rooting was obtained when subcultured shoots were preconditioned for 2 weeks in medium with 10 g l^–1 activated charcoal before treatment with 43 µm NAA or 39 µm IBA. Rooting was improved when galactose was used instead of sucrose in the rooting medium. The rooted plantlets were acclimatised successfully.Abbreviations NAA naphthaleneacetic acid - IBA indolebutyric acid - MS Murashige & Skoog Medium - WPM Woody Plant Medium - NN Nitsch Medium - Juv juvenile explant - Adu adult explant     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.