Endogenous indole-3-acetic acid and abscisic acid in apple microcuttings in relation to adventitious root formation

The effects of applying indole-3-butyric acid (IBA) for periods up to 48 h were examined in difficult-to-root microcuttings (from newly-established cultures) and in easy-to-root microcuttings (from long-term subcultures) of Jonathan apple (Malus X domestica Borkh). In easy-to-root material, 20% of the microcuttings produced roots in the absence of IBA, while 6 h exposure to 10 M IBA gave 100% rooting of microcuttings. In contrast, root formation in difficult-to-root material was IBA-dependent. Maximum rooting of these microcuttings (50%) required 24 h exposure to 10 M IBA.Variation in the endogenous levels of free indole-3-acetic acid (IAA) during the course of root induction was similar in microcuttings of both types but there were marked differences in endogenous abscisic acid (ABA) levels. In easy-to-root microcuttings ABA remained at a constant low level, but in difficult-to-root material ABA exhibited marked fluctuations and was present at higher concentrations than in easy-to-root microcuttings.     

Effect of culture media and irradiance level on growth and morphology of Persea americana Mill microcuttings

Avocado shoots were multiplied in vitro in two culture media of different consistency, double phase and solid medium, at three different irradiance levels: 35, 60 and 85 μmol m⁻² s⁻¹. Effects of culture and environmental conditions in multiplication rate, rooting capacity, hyperhydricity and leaf surface morphology of microcuttings were evaluated. Double phase medium induced hyperhydricity, producing leaf microcuttings with deformed stomata and low crystalline epicuticular waxes; microcuttings also showed reduced rooting capacity. By contrast, solid medium promoted leaf area development on microcuttings and decreased hyperhydricity. Stomatal index was not affected by these treatments but stomatal density was, interacting with the amount of irradiance applied. Increasing irradiance decreased concentration of chlorophyll a and carotenoids in the leaf but did not affect leaf hyperhydricity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange [<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osb. × <Emphasis Type="Italic">Poncirius trifoliata</Emphasis> (L.) Raf]

Due to widespread polyembryony, Citrus rootstocks are usually propagated by open-pollinated seed germination, although micropropagation offers many advantages. Encapsulation technology has recently attracted the interest of researchers in the field of plant propagation because it combines the advantages of zygotic or gamic seeds with those of micropropagation. In this study, we examined the encapsulation of Carrizo citrange uninodal microcuttings (3–4 mm long) and evaluated the influence of the calcium alginate coating, a short time storage at cold temperature, and different sowing substrates on the viability and regrowth of the explants. A secondary aim was to develop an efficient protocol to induce root formation in the microcuttings. The results showed that encapsulation did not negatively affect the viability, providing a satisfactory regrowth, and storage potential for 30 days at low temperature. No differences in viability and regrowth were detected between the two different sowing substrates tested (agar-solidified medium and paper filter). To optimize the production of microcuttings required to perform the encapsulation experiments, in a preliminary experiment we assessed different factors affecting the in vitro shoot regeneration from epicotyl segments (obtained from seeds of Carrizo citrange germinated in vitro), including the influence of in vitro organogenesis, explant orientation, cut surface contact with the medium, treatments with different growth regulators, and distance of the organogenic explants from the cotyledonary node. The highest organogenic response was obtained from segments horizontally cultured (particularly from the basal portion), from segments with the cut surface in contact with the medium and from explants cultured on medium supplemented with 6-benzyladenine. No significant difference in regeneration efficiency was found in response to the distance of the epicotyl portions from the cotyledonary node.     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

The influence of ethylene on in vitro rooting of GF 677 (Prunus persica × Prunus amygdalus) hybrid peach rootstock

Summary Treatments differing from each other for the type of tube closure (i.e., cotton plug for free gas exchange, airtight rubber cap, and rubber cap with ethysorb) and/or rooting culture medium (i.e., enriched or not by 25 to 100 μM acetylsalicylic acid) were compared for their effects on gaseous composition of the culture atmosphere and microcutting rooting of the GF 677 (Prunus persica × Prunus amygdalus) hybrid. Rubber capping, which leads to rapid ethylene accumulation inside tubes, strongly reduced rooting time and in some cases enhanced final rooting percentage over that of cotton plugs. Ethysorb almost completely absorbed ethylene produced by shoots, which showed lower rooting percentages within 9 d than microcuttings cultured in the absence of ethysorb. In contrast, no significant difference in rooting was found between the two treatments after 14 d. Carbon dioxide concentration was similar in all treatments within 5 to 9 d and seemed to be ineffective for rooting. The influence of acetylsalicylic acid on rooting was unclear. Root number and length were not significantly influenced by the treatments. These results demonstrate that the use of airtight closures, leading to rapid ethylene accumulation, can reduce time of rooting expression for GF 677 microcuttings. However, free gas exchange towards the end of the rooting period (from Day 9 to Day 14) is advisable to prevent leaf yellowing. No significant difference in plantlet survival and growth after transfer ex vitro was found among treatments.     

Role of auxin and its modulators in the adventitious rooting of <Emphasis Type="Italic">Eucalyptus</Emphasis> species differing in recalcitrance

It is well established that auxins play a central role in the determination of rooting capacity, which is essential for vegetative propagation. Recent studies with apple trees have pointed to significant effects of auxin stability, wound related phenolics and ethylene production in the control of adventitious rooting. In the present study, a comparative analysis of the adventitious rooting of microcuttings of Eucalyptus saligna (easy-to-root species) and Eucalyptus globulus (difficult-to-root species) was carried out with different types of auxins, light intensities, presence or absence of apical meristem, different concentrations of phenolic compounds and presence or absence of an ethylene action inhibitor. Parameters evaluated were the percent rooting, number of roots per rooted cutting, length of longest root and mean rooting time. Results showed that auxins of intermediate stability are more favorable to rooting (particularly for the recalcitrant species), higher light intensities in the presence of exogenous auxins promote the rooting response, the absence of meristematic apex or externally supplied phenolics are not limiting for the rooting induced by exogenous auxins, and ethylene appears to play a minor role in the development of adventitious roots in microcuttings of Eucalyptus, indicating that the rhizogenic response results from direct effect of auxins.     

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G₁ and IR-M₂ and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

Comparison of gene expression under <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">ex vitro</Emphasis> conditions during rooting of grape cuttings through mRNA differential display

An mRNA differential display (DD) analysis during rooting in grape cuttings was carried out to determine whether gene expression patterns differed under in vitro and ex vitro conditions. The four tissue samples for differential display and subsequent Northern hybridization analyses included control stem tissue from in vitro and ex vitro sources, microcuttings planted in MS based in vitro rooting medium and softwood cuttings planted in ex vitro soil medium, both collected 48 h after planting. DD autoradiographs showed gross similarity in banding pattern between in vitro and ex vitro stem tissue, whether constitutive or induced. Northern blot analysis of a few bands that appeared to be differentials did not indicate them as true positives. The results suggested that gene expression pattern during physiological processes such as rooting may be identical under in vitro and ex vitro conditions.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Comparative responses of the Savanna grasses Cenchrus ciliaris and Themeda triandra to defoliation

Summary Two perennial tussock grasses of savannas were compared in a glasshouse study to determine why they differed in their ability to withstand frequent, heavy grazing; Cenchrus ciliaris is tolerant and Themeda triandra is intolerant of heavy grazing. Frequent defoliation at weekly intervals for six weeks reduced shoot biomass production over a subsequent 42 day regrowth period compared with previously undefoliated plants (infrequent) in T. triandra, but not in C. ciliaris. Leaf area of T. triandra expanded rapidly following defoliation but high initial relative growth rates of shoots were not sustained after 14 days of regrowth because of reducing light utilising efficiency of leaves. Frequently defoliated plants were slower in rate of leaf area expansion and this was associated with reduced photosynthetic capacity of newly formed leaves, lower allocation of photosynthate to leaves but not lower tiller numbers. T. triandra appears well adapted to a regime where defoliation is sufficiently infrequent to allow carbon to be fixed to replace that used in initial leaf area expansion. In contrast, C. ciliaris is better adapted to frequent defoliation than is T. triandra, because horizontally orientated nodal tillers are produced below the defoliation level. This morphological adaptation resulted in a 10-fold higher leaf area remaining after defoliation compared with similarly defoliated T. triandra, which together with the maintenance of moderate levels of light utilising efficiency, contributed to the higher leaf area and shoot weight throughout the regrowth period.     

Dugong grazing and turtle cropping: grazing optimization in tropical seagrass systems?

Grazing by dugongs and cropping by green turtles have the capacity to alter the subsequent nutritional quality of seagrass regrowth. We examined the effects of simulated light and intensive grazing by dugongs and cropping by turtles on eight nutritionally relevant measures of seagrass chemical composition over two regrowth periods (short-term, 1–4 months; long-term, 11–13 months) at two seagrass communities (a mixed species community with Zostera capricorni, Halophila ovalis, Halodule uninervis, Cymodocea rotundata and C. serrulate; and a monospecific bed of Halodule uninervis) in tropical Queensland, Australia. The concentrations of organic matter, total nitrogen, total water-soluble carbohydrates, total starch, neutral detergent fiber, acid detergent fiber, acid lignin, as well as the in vitro dry matter digestibility (IVDMD) were measured in the leaves and below-ground parts of each species using near-infrared reflectance spectroscopy (NIRS). Regrowth of preferred species such as H. ovalis and H. uninervis from simulated intensive dugong grazing after a year exhibited increased (by 35 and 25%, respectively, relative to controls) whole-plant N concentrations. Similarly, regrowth of H. ovalis from simulated turtle cropping showed an increase in the leaf N concentration of 30% after a year. However, these gains are tempered by reductions in starch concentrations and increases in fiber. In the short-term, the N concentrations increased while the fiber concentrations decreased. These data provide experimental support for a grazing optimization view of herbivory in the tropical seagrass system, but with feedback in a different manner. Furthermore, we suggest that in areas where grazing is the only major source of natural disturbance, it is likely that there are potential ecosystem level effects if and when numbers of dugongs and turtles are reduced.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Retardation of tree growth by injection of plant growth regulators

Previously topped American sycamore (Platanus occidentalis L.), cottonwood (Populus deltoides Bartr. ex Marsh), water oak (Quercus nigra L.), and bigleaf maple (Acer macrophyllum Pursh) trees were injected with aqueous solutions of maleic hydrazide or dikegulac-sodium in April-June, 1979 at various locations around the United States. Measurements made later in the autumn showed that dikegulac-sodium reduced sprout regrowth in all four species, whereas maleic hydrazide was only effective in controlling regrowth of American sycamore and cottonwood. The effects of dikegulac persisted through the second growing season. Significant variability was observed among treated trees and must be reduced in order to achieve consistent growth responses from an application of growth retardants.This paper reports the results of research only. Mention of a growth regulator does not consitute a recommendation for use by the U.S. Department of Agriculture nor does it imply registration under FIFRA as amended. Mention of a trademark or propriety product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Influence of endophyte infection, plant age and harvest interval on Rhopalosiphum padi survival and its relation to quantity of N-formyl and N-acetyl loline in tall fescue

Rhopalosiphum padi L. (Homoptera: Aphididae) is sensitive to loline alkaloids present in tall fescue, Festuca arundinacea Shreb., infected with the endophytic fungus, Acremonium coenophialum Morgan-Jones & Gams. Aphid survival was higher on endophyte-free plants regardless of plant age after germination or age of regrowth tissue after clipping. Survival of aphids on endophyte-infected grass was lower on young tissue but increased as plants aged, although it never reached the same level on endophyte-free plants. Both N-formyl and N-acetyl loline increased as uncut or regrowth tissue aged; however, this was influenced by the age of the plant at the initial cut and the clipping frequency. Although even small amounts of loline cause high aphid mortality, the aphids are able to survive on endophyte-infected plants if the tillers have senescing leaves which contain lower amounts of loline. Preference for senescing leaves may help R. padi avoid plant parts containing high amounts of toxic allelochemicals, thus contributing to higher numbers of aphids on older, endophyte-infected plants.     

Influence of cytokinin levels on <Emphasis Type="Italic">in vitro</Emphasis> propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Soil and vegetation response to thinning White Cypress Pine (Callitris glaucophylla) on the North Western Slopes of New South Wales, Australia

Dense White Cypress Pine (Callitris glaucophylla J. Thompson and L.A.S. Johnson) regrowth occurs frequently across previously cleared landscapes in New South Wales (NSW), and is thought to adversely affect agricultural production and to cause land degradation. The NSW Native Vegetation Act (2003) requires that management of native vegetation including pre-1990 regrowth must ‘improve or maintain’ site condition, yet there is currently limited information regarding techniques for the optimum management of C. glaucophylla in this regard. We conducted a preliminary study to examine floristic composition, soil condition (to 50 cm) and carbon storage under ‘Dense’ (dense regrowth), ‘Thinned’ (dense regrowth thinned 2000/2001) and ‘Un-colonised’ (pasture not yet recolonised by C.␣glaucophylla) plots on private lands in NSW. Reduced tree density from thinning resulted in increased biomass of the remaining individual trees. Un-colonised plots had significantly more groundcover than thinned plots, which had significantly more groundcover than dense plots. Differences in plant diversity however, were explained by site factors rather than land use. Soils in the dense plots were the most acid but soil pH was significantly higher in thinned plots and pH was highest in soil of the un-colonised plots. Mean values for carbon, nitrogen, sulphur and extractable phosphorus varied among sites, although each were significantly more abundant in the mineral soil of dense and thinned plots compared with un-colonised plots, suggesting that thinning had had a minimal effect on the soil parameters assessed. Accounting for all site components, site carbon storage was significantly higher in dense and thinned plots compared with un-colonised plots due to elevated levels of soil and litter carbon as well as the presence of trees. The results indicate that thinning dense C. glaucophylla can maintain and (by some measures) improve site condition. However, given the variability in some of the parameters assessed, further study across a wider range of soil types and rainfall gradients is proposed.