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     

Is it possible to manipulate root anchorage in young trees?

The optimal root system architecture for increased tree anchorage has not yet been determined and in particular, the role of the tap root remains elusive. In Maritime pine (Pinus pinaster Ait.), tap roots may play an important role in anchoring young trees, but in adult trees, their growth is often impeded by the presence of a hard pan layer in the soil and the tap root becomes a minor component of tree anchorage. To understand better the role of the tap root in young trees, we grew cuttings (no tap root present) and seedlings where the tap root had (?) or had not (+) been pruned, in the field for 7 years. The force (F) necessary to deflect the stem sideways was then measured and divided by stem cross-sectional area (CSA), giving a parameter analogous to stress during bending. Root systems were extracted and root architecture and wood mechanical properties (density and longitudinal modulus of elasticity, E _L ) determined. In seedlings (?) tap roots, new roots had regenerated where the tap root had been pruned, whereas in cuttings, one or two lateral roots had grown downwards and acted as tap roots. Cuttings had significantly less lateral roots than the other treatments, but those near the soil surface were 14% and 23% thicker than plants (+) and (?) tap roots, respectively. Cuttings were smaller than seedlings, but were not relatively less resistant to stem deflection, probably because the thicker lateral roots compensated for their lower number. Apart from stem volume which was greater in trees (+) tap roots, no significant differences with regard to size or any root system variable were found in plants (?) or (+) tap roots. In all treatments, lateral roots were structurally reinforced through extra growth along the direction of the prevailing wind, which also improved tap root anchorage. Predictors of log F/CSA differed depending on treatment: in trees (?) tap roots, a combination of the predictors stem taper and %volume allocated to deep roots was highly regressed with log F/CSA (R ² = 0.83), unlike plants (+) tap roots where the combined predictors of lateral root number and root depth were best regressed with log F/CSA (R ² = 0.80). In cuttings, no clear relationships between log F/CSA and any parameter could be found. Wood density and E _L did not differ between roots, but did diminish with increasing distance from the stem in lateral roots. E _L was significantly lower in lateral roots from cuttings. Results showed that nursery techniques influence plant development but that the architectural pattern of Maritime pine root systems is stable, developing a sinker root system even when grown from cuttings. Anchorage is affected but the consequences for the long-term are still not known. Numerical modelling may be the only viable method to investigate the function that each root plays in adult tree anchorage.     

Endogenous Levels of Growth Regulators and Their Relationship to the Rooting of Dahlia Cuttings

The relationship between the level of endogenous growth regulators and the rooting ability of dahlia cuttings was investigated. Easy-to-root vegetative cuttings were compared with difficult-to-root, flower bud bearing cuttings and the easy-to-root cv. Choot Hashani was compared with the difficnlt-to-root cv. Orpheo. No differences in the level of extracted or diffusable auxin, nor in the level of rooting cofactors, were found between easy-to-root and difficult-to-root cuttings. No activity could be found at the R_f zone corresponding to Cofactor 4 which is considered the main rooting cofactor in other plants. Higher inhibitor activity levels (assayed by the wheat coleoptile test and the mung bean rooting test) were found in difficult-to-root than in easily rooted cuttings. Furthermore, the inhibitor activity level in exadates from decapitated plants, was higher in esodates from plants which bore difficult-to-root cuttings than from plants bearing easy-to-root ones. Grafting of an easy-to-root plant onto a difficult-to-root stock caused inhibition of rooting in the cuttings taken from the scion. Results suggest that inhibitors formed in the root system, move upwards and accumulate in the plant, there causing the inhibition of rooting. Evidence from chromatographic comparisons and exogenous treatments with ABA indicate that the main inhibitor involved in the inhibition of rooting in dahlia cuttings is not ABA.     

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.     

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     

Mesorhizobium sp. J8 can establish symbiosis with Glycyrrhiza uralensis,increasing glycyrrhizin production

Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO₃. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.     

Differences in endo/exogenous auxin profile in cuttings of different physiological ages

The process of physiological ageing in woody plants is a very important factor influencing adventitious rooting. However, there is a lack of knowledge of biochemical backgrounds triggering ageing and consequently, rhizogenesis. Experiments with Prunus subhirtella ‘Autumnalis’ leafy cuttings of three different physiological ages (adult (over 40-year-old stock plants), semi-adult (5-year-old cutting plants) and juvenile (5-year-old in vitro plants)) were conducted in 2009. Half of the cuttings were banded ca. 3 cm above the bottom of the cutting with aluminum wire prior to insertion into the substrate to block the polar auxin transport. IBA, which was exogenously applied to the cuttings, could only be detected in the base of the cuttings on the first day after severance. Juvenile cuttings tended to have the highest values, but the effect was age specific. Later, the detection was not possible, regardless of the age. The IAA profile in cutting bases was similar for all physiological ages, reaching the peak on the first day after severance. Juvenile cuttings, in which the stems had been banded before insertion, contained more IAA in their bases on day 1 compared to the stems, which were not banded. These cuttings presumably transported absorbed auxin mainly via phloem, and not via mass flow like semi-adult and adult cuttings, where IAA concentrations were similar or even greater in non-banded cuttings compared to banded ones. These cuttings also tended to exhibit the best rooting results. The IAA-Asp accumulation was especially strong in adult cuttings, which contained significantly more aspartate on the first and third days after severance when compared with semi-adult and juvenile cuttings.     

Evaluation of aeroponics for clonal propagation of Caralluma edulis,Leptadenia reticulata and Tylophora indica – three threatened medicinal Asclepiads

The present study explores the potential of aeroponic system for clonal propagation of Caralluma edulis (Paimpa) a rare, threatened and endemic edible species, Leptadenia reticulata (Jeewanti), a threatened liana used as promoter of health and Tylophora indica (Burm.f.) Merill, a valuable medicinal climber. Experiments were conducted to asses the effect of exogenous auxin (naphthalene acetic acid, indole-3-butyric acid, indole-3-acetic acid) and auxin concentrations (0.0, 0.5, 1, 2, 3, 4 or 5gl⁻¹) on various root morphological traits of cuttings in the aeroponic chamber. Amongst all the auxins tested, significant effects on the length, number and percentage of rooting was observed in IBA treated nodal cuttings. Cent per cent of the stem cuttings of C. edulis rooted if pre-treated with 2.0 gl⁻¹ of IBA for 5 min while 97.7 % of the stem cuttings of L. reticulata and 93.33 % of stem cuttings of Tylophora indica rooted with pre-treatment of 3.0 gl⁻¹ of IBA for 5 min. Presence of at least two leaves on the nodal cuttings of L. reticulata and T. indica was found to be a prerequisite for root induction. In all the species, the number of adventitious roots per cutting and the percentage of cuttings rooted aeroponically were significantly higher than the soil grown stem cuttings. Shoot growth measured in terms of shoot length was significantly higher in cuttings rooted aeroponically as compared to the cuttings rooted under soil conditions. All the plants sprouted and rooted aeroponically survived on transfer to soil. This is the first report of clonal propagation in an aeroponic system for these plants. This study suggests aeroponics as an economic method for rapid root induction and clonal propagation of these three endangered and medicinally important plants which require focused efforts on conservation and sustainable utilization.     

A relationship between irradiation, carbohydrates and rooting in cuttings of Pisum sativum

Rooting ability was studied for cuttings derived from pea plants ( Pisum sativum , L. cv. Alaska) grown in controlled environment rooms. When the cuttings were rooted at 70 μmol m⁻² s⁻, ¹ (photosynthetic photon flux density) or more, a stock plant irradiance at 100 μmol m⁻² s⁻¹ decreased rooting ability in cuttings compared to 5 μmol m⁻², s⁻¹, However, cuttings rooted at 160 μmol m⁻² s⁻¹ formed more roots compared to 5 (μmol m⁻² s⁻¹. Although a high irradiance increased the number of roots formed, it could not overcome a decreased potential for root formation in stock plants grown at high irradiance. Light compensation point and dark respiration of cuttings decreased by 70% during the rooting period, and the final levels were strongly influenced by the irradiance to the cuttings. Respiratory O₂ uptake decreased in the apex and the base of the cutting from day 2 onwards, whereas a constant level was found in the leaves. Only the content of extractable fructose, glucose, sucrose and starch varied during the early part of the rooting period. We conclude that the observed changes in the cuttings are initiated by excision of the root system, and are not involved in the initiation of adventitious roots.     

Influence of the Irradiance on Carbohydrate Content and Rooting of Cuttings of Pine Seedlings (Pinus sylvestris L.)

Seedlings of Pinus sylvestris L. were grown for 6 weeks at an irradiance of either 8 or 40 watts per square meter in a controlled environment room. Cuttings from these plants were rooted in tap water for 75 days at either 8 or 40 watts per square meter. The photoperiod was 17 hours.

During the first 30 days of the rooting period quantitative changes in carbohydrates were recorded in cuttings from the different treatments. The carbohydrate contents of the cuttings were mainly regulated by the irradiance during the stock plant stage and generally a higher carbohydrate level was found in cuttings from stock plants grown at 40 watts per square meter.

The irradiance during the rooting period had only minor effects on the time course of root formation, whereas the irradiance during the stock plant stage did influence the subsequent root formation. Cuttings from stock plants grown at 8 watts per square meter rooted faster and with higher frequency than those from stock plants grown at 40 watts per square meter. These results are discussed in relation to the mentioned irradiance effects on carbohydrate content.

     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

Growth Performance of Cuttings Raised from in vitro and in vivo Propagated Stock Plants of Rosa damascena Mill.

Comparative studies on rooting and growth performance of cuttings raised from in vitro and in vivo grown plants of Rosa damascena are described. Cuttings were treated with different auxins and upon transfer to soil their growth performance was recorded. Overall, the auxin treated cuttings of in vitro raised plants responded better than the cuttings of in vivo raised plants. Optimal response for percentage of rooting, root number, root length and bottom breaks was observed at 100 mg dm^–3 IBA. The cuttings derived from in vitro raised plants showed a significantly better response for percent rooting, root number, root length and bottom buds in control treatments.     

Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.Abbreviations BA Benzyl-Aminopurine - NAA Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - MS medium Murashige and Skoog's medium - DBH Diameter at Breast Height     

Effects of combinational treatment with ethephon and indole-3-butyric acid on adventitious rooting of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> cuttings

Adventitious rooting is essential for cutting propagation of pine wilt-resistant Pinus thunbergii. To examine a variety of adventitious rooting potentials among donor plants, cuttings were taken from 31 seedlings within a half-sib family. Rooting abilities of cuttings from each seedling ranged from 0 to 100%. When 11 ortets and 11 ramets (clonally propagated from each ortet) were used as donor plants, there was a positive correlation between rooting abilities of cuttings from ortets and ramets, suggesting that adventitious rooting is dependent on genetic factors in the donor plants. To promote adventitious rooting of cuttings by hormonal treatment, we examined the effect of soaking time in Oxyberon (19.7 mM indole-3-butyric acid (IBA) solution) on rooting. Ten minutes was the best soaking time for rooted cuttings to produce more adventitious roots without impairing normal growth. When cuttings were soaked in Ethrel diluent (69.2 μM ethephon) for 24 h before soaking in Oxyberon for 10 min, a significantly higher rooting ability was observed than those soaked in Oxyberon alone. Ethrel on its own barely affected rooting ability. The positive effect of the combinational treatment was confirmed in a similar experiment using authentic ethephon and IBA instead of Ethrel and Oxyberon. When cuttings were soaked in a mixture of ethephon and silver thiosulfate (STS), an ethylene action inhibitor, before IBA-soaking, the effect was partially diminished compared with combinational treatment without STS. These findings suggest that ethylene action caused by ethephon treatment promotes IBA-mediated adventitious rooting of P. thunbergii cuttings.     

GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta

A Geographical Information Systems (GIS)-facilitated approach for the in vitro propagation and ex situ conservation of the conservation priority species Achillea occulta is presented. To realize the species?? ecological requirements, the coordinates of the original habitat were linked with thematic layers derived from digital databases in a GIS environment. From wild plants, shoot tips were established in vitro in a basal medium with 4???M 6-benzyladenine (BA) and 0.5???M indole-3-butyric acid (IBA). A modified basal MS medium (modMS, double amount of Fe) proved to be the most effective for in vitro adventitious shoot production. The effect of BA in combination with ??-naphthaleneacetic acid (NAA) or IBA on shoot proliferation was also tested. The highest number of new microshoots/explant (3.5), with 0.93?cm shoot height was obtained when the modMS was supplemented with 5???M BA and 2.5???M IBA. To evaluate the root induction ability, microshoots produced were transferred to modMS media supplemented with 0?C20???M IBA and 0?C20???M NAA. Rooting proved to be very difficult and only by adding 20???M IBA, a 12.5% rooting percentage was achieved. Acclimatization succeeded only during early spring. Young plants transplanted at the Balkan Botanic Garden of Kroussia produced flowers and seeds in the first year. This GIS-approach provided useful guidelines for A. occulta??s (a) effective propagation (selection of greenhouse temperatures, temperatures during in vitro culture, suitable period for cuttings and acclimatization of plantlets), and (b) ex situ cultivation (selection of watering regime, temperatures, locations and exposures for growing sites).     

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.     

An analysis of responses of resistant and of susceptible tomato plants to Verticillium infection

Comparative studies were made of the responses of resistant and of susceptible Gem tomato plants to infection by Verticillium albo-atrum. When inoculated through roots, there were striking differences in their responses. In susceptible plants, the foliar symptoms and amount of mycelium in the stem increased rapidly for some time. Then the mycelium started to disappear from the stem; this was accompanied by a check to the normal progress of symptoms, and by the formation of tyloses. In resistant plants, a limited invasion of the root and lower stem was accompanied by rapid and extensive tylosis. The mycelium soon disappeared from the stem and the plant then recovered from the initially mild symptoms. There was an inverse relationship between the amount of mycelium and the extent of tylosis in infected plants. The growth of susceptible plants was markedly reduced by infection. Total leaf area was much less because the newly produced leaves did not expand normally. The root system in infected plants was smaller because there were many fewer tertiary roots. In resistant plants infection stimulated growth. Tomato cuttings inoculated with conidia reacted similarly to root-inoculated plants. Hyphae grew well in the vascular system of susceptible cuttings whereas in resistant cuttings the pathogen started to grow but soon disappeared. Detached leaves of susceptible plants, inoculated through cut ends, wilted more than did leaves from resistant plants. It is suggested that resistance is mainly of the active type that develops after infection.     

Dynamics of extractable carbohydrates in Pisum sativum. II. Carbohydrate content and photosynthesis of pea cuttings in relation to irradiance and stock plant temperature and genotype

Rooting ability was studied for cuttings derived from stock plants of wild type pea seedlings and seedings of two mutants deficient in photosystem II activity and chlorophyll. Stock plants were grown at 15, 20, 25 or 30°C at 38 W m^-2. Cuttings were rooted at 20°C and at an irradiance of 16 or 38 W m^-2. The rooting ability seemed to be correlated with the initial carbohydrate content only at 38 W m^-2. Based on the findings of the present study it may be concluded that for pea seedlings the growth temperature is more important than photosynthesis as regards accumulation of extractable carbohydrates. During the rooting period carbohydrates are necessary for root formation, but the effect of the iradiance on the number of roots formed is not mediated by the carbohydrate content. Under specific rooting conditions it is possible to correlate the initial carbohydrate content with the rooting capacity of the cuttings within a phenotype, but not always when different phenotypes are considered. The results indicate a connection between the metabolic activity of the cuttings and their ability to form adventitious roots.