Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1–50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.Abbreviations 2ip N-isopentenyl adenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MES 2-(Nmorpholino)ethane acid - NAA naphthaleneacetic acid - TDZ thidiazuron - BAP 6-benzylaminopurine - PEG polyethylene glycol     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

Potential use of new diphenylurea derivatives in micropropagation of Capparis spinosa L.

A protocol for in vitro multiplication of caper (Capparis spinosa L. subsp. rupestris) from nodal segments collected from mature plants was developed. For shoot multiplication, one auxin (indol-3-butyric acid, IBA) and cytokinins of two different classes were used: the N6-substituted adenine derivatives 6-benzylamino purine (BAP), and the two synthetic phenylurea derivatives N-phenyl-N′-benzothiazol-6-ylurea (PBU) and N-phenyl-N′-(1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ). Maximum shoot production was achieved from explants cultured with the adeninic cytokinin BAP (4 μM) and the auxin IBA (0.5 μM). New shoots longer than 1 cm were used for rooting. To induce root formation, three auxins [indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA) and 3-Indoleacetic acid (IAA)] and two synthetic phenylurea derivatives [N,N-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU)] were used. All rooting compounds tested stimulated the formation of roots. However, the best result in terms of a high percentage of rooted shoots having a well-developed root system with many lateral roots was achieved with the synthetic phenylurea 2,3-MDPU (1 μM) with 93.7% of well rooted plantlets. About 80% of rooted plantlets were successfully acclimatized and transferred to the greenhouse.     

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.     

Morpho-anatomical and biochemical changes associated with rooting of micropropagated ninebark cuttings

Ninebark (Physocarpus opulifolius) is an attractive ornamental shrub with poor rooting characteristics in some cultivars, which is a limiting factor in commercial production This study was designed to optimize rooting conditions of ninebark cuttings and to observe the effect of exogenous auxin IBA on some morpho-anatomical and biochemical changes associated with rhizogenesis in the in vitro conditions. Both auxins under study: the indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) gave comparable effects but the combination of ½ MS?+?1 mg·L^?1 IBA was the most cost effective for all rooting parameters. Anatomical changes at the cuttings’ bases during root formation were typical for woody plants and they were accelerated by auxin in the culture medium. High levels of the endogenous indole acid and hydrogen peroxide were temporarily associated with intensive cell divisions in cuttings, and the polyphenolic acid contents kept increasing during rooting above the initial levels and those in controls.

     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Shoot RNA, cambrial activity and indolebutyric acid effectivity in seasonal rooting of juvenile and mature Ficus pumila cuttings

The seasonal influence on adventitious root formation was studied in woody leaf bud cuttings of Ficus pumila L., creeping fig. Juvenile cuttings rooted easily, whereas only mature cuttings treated with indolebutyric acid (IBA) exceeded 30% rooting. Greater rooting occurred in IBA-treated juvenile and mature cuttings than controls, regardless of the month each experiment was initiated. Seasonal changes influenced rooting in all treatments except IBA treated juvenile cuttings where percentage rooting was not affected. Higher vascular cambial activity and shoot RNA levels occurred in juvenile and mature forms during peak rooting periods. Highest RNA was recorded with juvenile materials during maximum rooting periods, while lowest RNA was observed in mature shoots during low rooting intervals.     

Adventitious rooting performance in micropropagated Cornus mas

Axillary buds sampled from a mature 27-year-old Cornus mas cv. Macrocarpa were grown in vitro on modified woody plant medium (WPM). Adventitious rooting performance of microshoots was assayed on half-strength WPM supplemented with 1-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) under various pH. NAA induced significantly higher rooting frequencies than IBA. The pH of 6.8 inhibited rooting, and differentiated roots were extremely thick and fragile. The highest rooting frequency was recorded on half-strength WPM supplemented with 5.37 µM NAA at the pH value adjusted to 6.2 (73 % of rooted shoots). In the presence of IBA, the formation of adventitious roots was observed only in the basal part of the microshoot dipped into rooting medium. In the case of NAA, however, adventitious roots arose also from the parts of microshoots that were not in contact with medium. The growth of aerial roots was always positively gravitropic. The nuclear microsatellite Cf-G17 gave a monomorphic fingerprinting pattern across the mother shrub and micropropagated plantlets. Acclimatized plants did not show any visually detectable morphological variation and the aerial adventitious root formation was no longer observed.     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

Exogenous polyamines improve rooting of hazel microshoots

A strong positive effect of polyamines on rooting of microshoots of adult hazel (Corylus avellana L., cv. Gironell) is described. The effect of polyamines, both in the root induction solution and in the actual rooting medium, was assessed in order to study the effect on the successive rooting phases. Polyamines improved rooting of indole-3-butyric acid-treated microshoots in a synergistic fashion, perhaps by favouring a better induction of roots, with an acceleration of the response (only half the time required for rooting compared to the control). When applied without indole-3-butyric acid, polyamines had only a limited positive effect on rooting, although longer exposure times and/or higher concentrations could increase their effect. Possible rapid uptake and translocation of polyamines in the xylem in our system is discussed. The results offer a new approach to enhance rooting ability of species that are normally difficult to root.Abbreviations BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Changes in peroxidase activity,auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloïdes) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA naphthaleneacetic acid - IAA indole-3-acetic acid - 2-iP 2-isopentenyladenine - IAAsp indole-3-acetylaspartic acid - IBA indole-3-butyric acid - GC gas-chromatography     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

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.     

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

In vitro regeneration of Norway maple (Acer platanoides L.)

Regenerants were produced from axillary buds, but not from petiole segments, greenwood cuttings and leaf discs. Petiole segments and greenwood cuttings responded by massive callus cell proliferation without adventitious shoot formation. The development of induced buds into shoots occurred on WPM medium containing kinetin. Vigorous shoots larger than 2.0 cm in length were successfully rooted in half strength WPM medium supplemented with 1.0 mg dm^-3 indole-3-butyric acid.     

Nauclea diderrichii: rooting of stem cuttings,clonal variation in shoot dominance,and branch plagiotropism

Summary Nauclea diderrichii (De Wild, and Th. Dur.) Merill (Rubiaceae), an indigenous hardwood of West Africa, is increasingly being grown commercially. This study investigates the potential for vegetative propagation and clonal selection, and raises some fundamental questions about the physiology of apical dominance and of plagiotropism. Rooting ability was high, with up to 100% rooting in 2–4 weeks, when different Indole-3-butyric acid (IBA) concentrations and leaf areas were tested. Auxin applications greatly increased the numbers of roots per cutting. The decapitation of unbranched plants revealed clonal variation in apical dominance and also in the establishment of outright dominance by the two shoots formed from the outgrowth of the axillary buds of the opposite leaves at the top node. Regression analysis of the Dominance Ratio (length of dominant: length of the sub-dominant shoot at the time of achieving dominance) against overall lateral bud activity (r = 0.82), showed that when the two top shoots co-dominate they provide a more powerful source of Correlative Inhibition than when one of the top shoots dominates the other. The imposition of plagiotropism in the axillary bud occurred over a period of a few days as the terminal and axillary buds emerged from the stipule. Growth of accessory buds on intact plants and debranched cuttings was orthotropic. These results are discussed with regard to the role of the leaf in root formation and the understanding of dominance relationships, branching and crown development in trees.