The effect of triacontanol on micropropagation of balm, Melissa officinalis L.

 Triacontanol, a long-chain primary alcohol was found to be an effective growth regulator in the micropropagation of balm, Melissa officinalis. In both the multiplication and the rooting phase, concentrations of 2, 5, 10 and 20 μg triacontanol per liter were applied. After 4 weeks of culture, the fresh weight of shoots was measured in the multiplication phase and root formation, photosynthetic activity, chlorophyll content and the fresh and dry weights of shoots were analyzed in the root induction phase. In the multiplication phase, 5 μg/l triacontanol was found to be the optimal concentration, while in the rooting phase 2 μg/l was the most effective. Triacontanol increased the number and length of roots, and it enhanced shoot growth, fresh weight, and the chlorophyll content, but it had no effect on the dry weight and the photosynthetic activity of the plants. Results of our work demonstrate that triacontanol can be applied as an effective growth regulator in the tissue culture of balm. Received: 3 December 1997 / Revised: 24 February 1998 / Accepted: 26 February 1999     

Micropropagation of Bupleurum fruticosum: The effect of triacontanol

A micropropagation protocol of Bupleurum fruticosumL. was developed, in order to obtain a great number of plants for the production of secondary metabolites. The combination of 1.0 mg l^–1 indole-3-acetic acid and 1.5 mg l^–1 6-benzyladenine added to Murashige–Skoog medium resulted in the best multiplication. Root formation gave the same results in hormone-free medium and in the medium to which various concentrations of 1-naphthaleneacetic acid had been added. In both the multiplication and the rooting phase, 2, 5, 10 and 20 g l^–1 triacontanol were applied. After 4 weeks of culture, the number of shoots and nodes and the fresh weight were measured in the multiplication phase. Root number, shoot length, node number and fresh weight were determined in the root induction phase, while chlorophyll content was measured in both phases. In the multiplication phase 2 g l^–1 triacontanol was found to be the optimal concentration, the same as was the case in the rooting phase, except for the production of epigeous structures, for which the optimal concentration was 10 g l^–1.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

Rapid micropropagation of mature wild cherry

Explants taken from the mature vigorous tree of wild cherry (Prunus avium L.) were assayed for their organogenic capacity under various phytohormonal treatments. The highest rate of adventitious shoot multiplication was recorded at a combination of 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ thidiazuron (6.83 shoots per explant). No differences in multiplication rates were found among media supplemented with BAP, BAP + α-naphthaleneacetic acid (NAA) or BAP + indole-3-butyric acid (IBA). Shoot elongation was significantly affected by the concentration of BAP, regardless of auxin addition to medium. Up to 73 % of microshoots rooted after using 0.3 mg dm⁻³ IBA, otherwise the adventitious rooting occurred at reasonable frequencies in all auxin treatments. Regenerated plantlets were successfully hardened ex vitro and continued to grow after the transfer to soil. No morphological aberrations were observed in the regenerates.     

Rhizogenesis in Forsythia×intermedia and Syringa vulgaris; application of a simple internode experimental system

 Differences in rhizogenesis between easy-to-root Forsythia×intermedia Zab. cv. Lynwood and difficult-to-root Syringa vulgaris L. cv. Madame Lemoine were measured in an experimental system based upon internodal stem sections excised from axillary shoot cultures. Root induction in Syringa was distinctly polar, responding best to distal application of indole-3-butyric acid (IBA), whereas Forsythia was equally responsive to IBA applied at either end. Root initiation in Syringa declined rapidly from 73% to 32% over 48 h when the application of a 24-h pulse of IBA was delayed following excision of the internode; in Forsythia a smaller decline (93–70%) occurred over 144 h. Forsythia internodes were the more responsive, and developed roots after distal or proximal application of 3 μM IBA, whereas Syringa required the distal application of 30 μM IBA. Received: 22 March 2000 / Revision received: 5 June 2000 / Accepted: 5 June 2000     

Micropropagation of Ceratopetalum gummiferum, an important Australian cut flower corp

Summary Ceratopetalum gummiferum Sm. ‘Albery's Red’ (NSW Christmas Bush), a native to eastern Australia, has become an important commercial plant in both the export and domestic markets. A protocol for in vitro culture was investigated for rapid clonal propagation of selected cultivars. Murashige and Skoog medium, supplemented with various concentrations of 6-benzylaminopurine, kinetin, 6(γ,γ-dimethylallylamino)-purine or zeatin were examined for their effects on multiplication. The most successful treatments were 2.2 μM 6-benzylaminopurine and 11.6 μM kinetin which increased shoot number and explant weight. Although zeatin and 6(γ,γ-dimethylallylamino)-purine increased shoot length, both failed to increase multiplication rates. However, hyperhydricity was found to be a serious physiological disorder in tissue culture of C. gummiferum ‘Albery's Red’. Rooting in vitro was also examined with indole-3-butyric acid and naphthalene acetic acid, the most successful being 4.9 mM indole-3-butyric acid. The development of an in vivo rooting protocol, however, may prove to be essential for the commercial production of this plant.     

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     

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     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Use of sugarcane bagasse as an alternative low-cost support material during the rooting stage of apple micropropagation

Summary Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.     

Interaction of explant type and indole-3-butyric acid during rooting in vitro in a range of difficult and easy-to-root woody plants

Axillary shoots of woody species that were easy-to-root (Forsythia × intermedia, Betula pendula and juvenile Quercus robur) and difficult-to-root (Syringa vulgaris, ‘intermediate’ phase Q. robur and Daphne cneorum), were used to obtain various explant types (Entire, Detipped, Nodal or Internodal) that differed in their rooting responses. When apices or axillary buds were removed from explants, rooting levels decreased, and the time to root and loss of synchrony of rooting increased. Exogenous indole-3-butyric acid generally overcame these trends, and was essential for root induction in ‘intermediate’ Quercus and Daphne, and in all internode explants. The interaction between explant type and indole-3-butyric acid suggests a role for endogenous factors in the initiation and development of rooting. The internode explant has potential as a tool for further physiological and biochemical studies, as it is auxin-dependent and provides rooting-permissive or non-permissive states. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

The effect of paclobutrazol on in vitro rooting,transplant establishment and growth of fruit plants

The effect of paclobutrazol on in vitro rooting and growth of sour cherry (Prunus cerasus) rootstock CAB 11E clone, of S 749 × S 1490 (Prunus persica × Prunus kansuensis) hybrid rootstock, and of pear (Pyrus communis), cv. Abbé Fetel is reported.PP333 increased rooting of S 749 × S 1490 and of Abbé Fetel, particularly at a concentration of 0.5 mg/l (a.i.); moreover, it induced a rooting percentage as high as auxin in the former and hastened rooting of the latter. By contrast, paclobutrazol did not affect root production of 11 E.PP333-treated plants had shorter and thicker roots than controls but similar survival rates during acclimatization. Otherwise they grew less than controls during the first part of the acclimatization phase.Abbreviations used in text and tables BA = 6-benzyladenine - IBA = indole-3-butyric acid - PP333 = paclobutrazol = (2RS,3RS)-1-(-4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol Part of the results referring to S 749 × S 1490 (P. persica × P. kansuensis) rootstock were presented at the meeting on Controllo della fruttificazione delle piante da frutto, Bologna, Italy, June 1986, and were published in the Riv. Ortoflorofrutt. It. 70 (6)(1986). This research was funded in part by the Italian Ministry of Education (M.P.I. 60%).     

In vitro regeneration of loblolly pine and random amplified polymorphic DNA analyses of regenerated plantlets

 Adventitious buds were induced from organogenic callus derived from mature zygotic embryos of three lines (E-311, E-440, and E-822) of loblolly pine (Pinus taeda L.) within 27 weeks of culture. The influence of cytokinins, silver nitrate, and low-temperature treatment on the differentiation of adventitious buds was analyzed. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). After adventitious shoots had rooted on TE medium supplemented with 0.5 mg/l IBA, 2 mg/l BA, and 0.5 mg/l gibberellic acid, 498 regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1 :>: 1) soil mixture; 351 of these survived in the field. Total DNA was extracted from 21 regenerated plantlets randomly chosen from the 151 regenerated plantlets of line E-822. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed that 21 primers gave 107 clear reproducible bands, with the amplification products being monomorphic for all of the plantlets of line E-822 tested. A total of 2,247 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested plantlets. These results suggest that somatic organogenesis can be used for clonal micropropagation of some lines of loblolly pine without the fear of the appearance of unwanted somaclonal variants. Received: 5 August 2000 / Revision received: 5 September 2000 / Accepted: 10 October 2000     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of ‘Durondeau’ pear in modified-gelled medium

Summary The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs. extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an, increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.     

Juvenile stockplant material enhances root development through higher endogenous auxin level

Stockplants with various physiological characteristics were compared in a propagation experiment with leafy cuttings of ornamental cherry Prunus subhirtella ‘Autumnalis’. At the time of severance, cuttings harvested from juvenile 3-year-old in vitro-derived plants contained in their bases nearly twice as much IAA (indole-3-acetic acid) as cuttings derived from mature 40-year-old trees. Juvenile cuttings showed better rooting success in the propagation season. They developed a significantly higher number of primary roots and grew better than physiologically older cuttings. IAA time-course levels in cutting bases in the days after severance were similar in both cuttings types. They decreased over the first day (rooting late initiation phase) after severance until the third day after severance when the levels increased again (rooting induction phase and beginning of the root developing phase). At the time of severance, juvenile cuttings also contained higher concentrations of IAAasp (indole-3-acetyl aspartic acid) in their bases than mature cuttings. IAAasp time-course levels were similar to those measured for IAA.     

The effect of triacontanol on micropropagation and on secretory system of Thymus mastichina

Shoot multiplication of Thymus mastichina L. was achieved on media containing 0.1 mg l^–1 6-benzyladenine and/or 0.1 mg l^–1 indole-3-butyric acid, or in hormone-free medium (control). The growth of plantlets, the production and composition of the essential oil, the density and secretory stage of glandular hairs have been evaluated in the presence and absence of growth regulators and triacontanol. We observed a positive effect of triacontanol on the growth of micropropagated plantlets using different conditions. Media with different levels of BA, IBA and TRIA resulted in no differences in the composition of the essential oil produced by plantlets. The major components of the oil were 1,8-cineole and linalool. An increase in the oil yield was observed especially when triacontanol was added to hormone-free medium. There was no correlation between changes in the oil yield and glandular hairs density, but the yield was dependent on the secretory stage of the glands.     

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.     

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.