Micropropagation of juvenile and adult Quercus suber L.

This paper describes research on the application of tissue culture techniques to the micropropagation of cork oak (Quercus suber L.), a forest species of ecological and industrial importance in the Mediterranean area. Apical buds and nodal stem segments were employed as initial explants. Their origins were young seedlings, stump sprouts and sprouts formed on cuttings collected from old trees.The action of the mineral medium and growth regulators was studied in the multiplication stage. Media with low concentrations of ions, such as Sommer's or Heller's, are more suitable for growth and proliferation of explants than other media richer in salts. It was also observed that cytokinin (BA) must be present for the culture development. Adding low concentrations of auxin (NAA) to the medium improves the multiplication rate, especially in vegetative material of adult origin.The auxin type is the most important factor in the promotion of rhizogenesis. The method of application determines the quality of the root system. Treatment with low concentrations of IBA added to the rooting medium gives the best results.High sucrose concentration also improves rooting. Diluting the mineral rooting medium is slightly favourable, although there is no significant difference between it and the standard mineral concentration.Abbreviations D Durzan's - GD Gresshoff & Doy's - H Heller's - L Lepoivre's - MS Murashige & Skoog's - SH Schenk & Hildebrandt's - S Sommer's medium     

Micropropagation of Frazinus angustifolia from mature and juvenile plant material

Micropropagation of Fraxinus angustifolia Vahl has been successfully achieved both from mature and juvenile plant material using shoot tip and nodal explants. Several basal media supplemented with benzyladenine (BA) and indolebutyric acid (IBA) were tested for shoot proliferation. The most new explants per mature explant (5.3) was obtained on DKW medium plus 4.4 M BA+0.98 M IBA. The most new explants per juvenile explant (5.6) was produced on QL medium plus 8.9 M BA+0.49 M IBA. Rooting was achieved on WPM supplemented with 0.98–4.9 M IBA. Rooted plantlets were transferred to soil and acclimatized with 85% survival.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA 1-naphthaleneacetic acid     

Micropropagation of Ilex aquifolium L.

Summary Two procedures were tested for micropropagation of Ilex aquifolium (English holly), one in which shoots proliferated on solid medium and another one using liquid medium. Different growth regulator treatments and supports were analyzed for optimizing in vitro rooting, showing that indolebutyric acid and agar or cellulose plugs gave the best results. The surival percentage of successfully in vitro or ex vitro rooted plants did not differ significantly between the best treatments. However, the efficiency with ex vitro rooting was 80%, while for in vitro rooting, the final efficiency was 64%. The results show that a correct manipulation of Murashige's stage II of micropropagation and eliminating or decreasing stage III are useful tools to reduce the requirements of acclimatization.     

Micropropagation of juvenile and mature american beech

The purpose of this study was to micropropagate juvenile and mature American beech (Fagus grandifolia Ehrh.) resistant to beech bark disease. Shoot tips (from juvenile seedlings and root sprouts of mature trees) and buds from branches of mature trees, were cultured and multiplied on aspen culture medium supplemented with 0.89 μM 6-benzyladenine, 0.27 μM α-naphthaleneacetic acid, 20 g l^-1 sucrose, and 7 g l^-1 Difco Bacto-agar. Rooting of shoots was achieved using a 1 minute dip in 12.3 mm indole-3-butyric acid, followed by culture in a Horticube saturated with half-strength aspen culture medium containing 20 g l^-1 sucrose. Thirteen of 33 mature genotypes were established from shoot tips taken from root sprouts, and six of 41 were established from mature buds. Plantlets of six genotypes were successfully acclimatized to a growth room for three months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of carbon source and concentration on development of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.) shoots cultivated <Emphasis Type="Italic">in vitro</Emphasis> from nodal explants

Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl⁻¹. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The best response was afforded by sucrose at 20 gl⁻¹ both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and in ‘NL1’ at 10 gl⁻¹ it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse.     

Micropropagation procedures for Leontochir ovallei

Leontochir ovallei Phil., an endangered Chilean species in the Alstroemericeae, was micropropagated on Murashige & Skoog medium supplemented with 4 M benzyladenine, 1 M indolebutyric acid and 146 mg l^-1 glutamine. Over 88% of the shoots rooted in vitro when treated with 10 M naphthaleneacetic acid and micropropagated plantlets were successfully transplanted into the greenhouse.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

Micropropagation of Eucalyptus nitens maiden (Shining gum)

Summary Eucalyptus nitens Maiden (shining gum) is a frost-tolerant species of Eucalyptus that can be used as an alternative species to Eucalyptus globulus in some regions of Portugal where winter temperatures are too low. Seedlings and 1-yr-old shoot tips and nodes were used for micropropagation of E. nitens. The best multiplication rate (2.25) was obtained when seedling shoots (<15 mm) were cultured on a medium containing the major nutrients (at half-strength) and minor elements of Murashige and Skoog (1962) medium, the organics of De Fossard medium (De Fossard et al., 1974) and a combination of benzyladenine (0.9 μM) and 1-naphthaleneacetic acid (0.05 μM). Seedling cuttings (4-,8-, and 10-wk-old) rooted well on media containing several concentrations of 3-indolebutyric acid (4.9, 9.8, and 14.8 μM) or 3-indoleacetic acid (5.7, 11.4, and 17.1 μM), giving frequencies of root induction above 80%. With this type of explant, root formation was also found on basal medium without growth regulators. Rooting of in vitro-propagated shoots obtained from seedlings (8-wk-old) after four subcultures (every 3 wk) was more difficult, with the best results obtained on a medium containing 14.7 μM 3-indolebutyric acid (60.0% root induction). No root formation was achieved when shoots from 1-yr-old explants were used. After a period of 4 mo., 96.3% of the plants transferred to the greenhouse survived acclimatization.     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of Rosmarinus officinalis L.

Single-node stem segments of Rosmarinus officinalis L. var. genuina forma erectus proved better explants than shoot tips (ca. 2 cm long) for extablishment of field-grown plants in aseptic cultures. Benzylaminopurine was far more effective than kinetin for shoot induction in shoot tips excised from aseptically-grown plants. Maximum numbers of shoot buds (ca. 14) were formed per explant at 0.2 mgl^-1 benzylaminopurine in 30 days. After further growth of isolated shoots and treatment with 0.25 mgl^-1 indolepropionic acid for 7 days, 80% shoots produced roots. In vitro raised plantlets were successfully grown in soil to plants. About 5,000 plants could be produced from a single nodal segment in 1 year.NBRI Research Publication No. 195 (N.S.)     

Micropropagation of adult Stone Pine (Pinus pinea L.)

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees. Paloma Moncaleán and Ricardo Javier Ordás contributed equally.     

Anti-Auxin Enhance Rosa Hybrida L. Micropropagation

Shoots of rose cultivars Super Star and Sonia were multiplied for ten subcultures at 4-week intervals on solidified Murashige and Skoog's medium supplemented with 22.19 M benzylaminopurine + 1.07 M napthalene acetic acid + 0.05 M gibberellic acid. Addition of anti-auxins 2,3,5-triiodobenzoic acid (TIBA; 2.0 M) and 2,4,6-trichlorophenoxy-acetic acid (2,4,6-T; 0.39 M) into proliferation medium increased number of shoots per explant and length of shoots in both cultivars. Treatment with TIBA increased also number of leaves per shoot and leaf chlorophyll content.     

Micropropagation of juvenile and adultDigitalis obscura and cardenolide content of clonally propagated plants

Summary Cultures ofDigitalis obscura L. were established from axillary buds of mature plants or leaves of seedlings obtained under aseptic conditions. Explants were cultured on Murashige and Skoog medium containing benzyladenine and/or naphthaleneacetic acid. Shoot proliferation from axillary buds was not affected by seasonal fluctuations in the stock plants and increased relative to the cytokinin concentration, but auxin reduced the multiplication rate. Differentiation of somatic embryos and adventitious buds from cultured leaves required naphthaleneacetic acid alone or combined with benzyladenine, respectively. Cardenolide pattern and content of the regenerated plants were determined by high performance liquid chromatography and radioimmunoassay, respectively. Several cardenolides of series A and C were identified in the regenerants; no significant differences were found in the cardenolide patterns. Digoxigenin derivatives were found in all clonally propagated plants, but the amount of these glycosides was much higher in those obtained from axillary buds. This is the first report on micropropagation ofD. obscura from mature plants. The financial support of CICYT, Madrid, Spain (project no. PB89-0419) is gratefully acknowledged.     

Micropropagation ofPrunus mume

Shoot proliferation from axillary buds ofPrunus mume Sieb. et Zucc. was obtained on Woody Plant Medium (WPM) supplemented with 1 to 5 M benzyladenine, 3% sorbitol and solidified with 0.5 to 0.7% agar. Effects of different carbon sources on shoot proliferation were examined. Glucose provided better shoot proliferation than sucrose, sorbitol and fructose. In the presence of sucrose, leaf chlorosis occurred and shoots gradually declined. Best rooting percentage was obtained on WPM supplemented with 1 M naphthaleneacetic acid. Rooted plantlets were acclimatized under intermittent mist. However, survival rate was relatively low (20 to 30%).     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

Micropropagation of nodal explants from adult trees of Cleistanthus collinus

The micropropagation of adult Cleistanthus collinus was accomplished. The nodal segments from terminal twigs of a 15-year-old tree and basal sprouts of a comparable chronological age were used for initiating shoot bud cultures. Washing explants with sterile mixtures of citric acid (520.5 μM) and PVP 40 (3.75 μM) three to four times controlled the leaching of brown inhibitory substances into the establishment medium. Axillary shoots proliferated best on MS medium containing citric acid (104.1 μM), and PVP 40 (12.5 or 25 μM) supplemented with 0.44 μM BA. The number of new shoots from nodal segments of explants placed on MS medium supplemented with 0.44 μM BA increased when the remaining lengths of nodal segments were transferred to fresh medium after the longer microshoots were harvested. The microshoots derived from basal sprouts rooted best (50%) when treated with 11.4 mM IAA for 2 min, whereas only 40% of the microshoots derived from terminal twigs produced roots after a 2-min exposure to 28.5 mM IAA. The placement of BA-soaked agar cubes on the apex-decapitated shoots controlled shoot-tip necrosis considerably. In general, explants from basal sprouts were more suitable than terminal twig explants for the micropropagation of adult trees of C. collinus. Received: 26 February 1997 / Revision received: 13 September 1997 / Accepted: 29 September 1997     

Micropropagation of <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>: a multipurpose tree

An in vitro propagation protocol has been developed for Hagenia abyssinica using original material from both juvenile and mature trees. Juvenile explants were obtained from seedlings, as well as shoots and meristems from 5 to 7-month-old greenhouse grown plants. Shoots collected from stem bases of five genotypes were used to establish cultures from mature trees. Explants of seedling origin were used to optimize the multiplication medium and growth regulators concentration. The best result was obtained from shoots subcultured on either MS or WPM medium supplemented with 4.4 M BAP and 0.49 M IBA. The initiated shoots from all the different explants were multiplied on these media. Rooting of shoots was achieved using MS medium containing macronutrients at one-third strength supplemented with 4.9 M IBA. The shoots were kept in the dark for 4 days and transferred to medium of the same composition but containing 0.3% activated charcoal without growth regulators. Up to 100% rooting was achieved depending on genotype. Shoots multiplied on MS medium rooted better than those multiplied on WPM. Plantlets were transferred to pots containing a mixture of soil and perlite in a 2:1 ratio, respectively, and were maintained in the greenhouse. Increased irradiance reduced stem and leaf lengths and increased branch number of micropropagated plants.     

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.     

Micropropagation of adult avocado

A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock. Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in Murashige and Skoog solid medium with macroelements at half strength and a 1.3 μM benzyladenine supplement. To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt formulation and 1.3 μM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements, 2.8 μM in the solid phase and 0.4 μM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 μM indolebutyric acid, followed by transfer to solid medium in the absence of auxin but with 1 g l⁻¹ activated charcoal. Survival rate during the acclimatization in the greenhouse was 70%. This revised version was published online in June 2006 with corrections to the Cover Date.