In vitro propagation of Populus x wilsocarpa — a hybrid of ornamental value

Populus x wilsocarpa, a hybrid of important ornamental value, cannot be seed-propagated, nor grafted, since a compatible rootstock has not been identified. A micropropagation protocol consisting of a series of steps was therefore developed to facilitate the commercial production of this species. The technique involved the transfer of swelling buds to a growth initiation medium with the following composition: N6 macronutrients, MS micronutrients and vitamins supplemented with 0.5 mg l^-1 BAP. The best buds were from dormant twigs, stored at 0–2°C and then forced to burst prior to culture initiation. Shoot multiplication was on a basal WPM medium including 0.1 mg l^-1 BAP and 0.001 mg l^-1 NAA. Shoot elongation and rooting was also on a basal WPM medium supplemented with 1.0 mg l^-1 GA₃ followed by a transfer to a peat-perlite mix in the greenhouse.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid (GA₃) - MS Murashige and Skoog [17] - NAA naphthaleneacetic acid - N6 medium [Chu et al., 7] - WPM woody plant medium [16]     

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.     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Pre and post-severance effects of light quality on carbohydrate dynamics and microcutting adventitious rooting of two Eucalyptus species of contrasting recalcitrance

Adventitious rooting is a complex developmental response affected by genetic and environmental factors. Radiation quality effects on adventitious rooting depend on characteristics such as species, growth stage, irradiance, spectral quality, and time of exposure. Eucalyptus is an essential genus for the paper industry, and high yield plantations depend on adventitious rooting of selected genotypes. This work addressed two hypotheses: (1) radiation quality equally affects adventitious rooting in Eucalyptus species of different recalcitrance; (2) adventitious rooting outcome depends on both donor plant and cutting radiation quality treatments. To that end, the easy-to-root Eucalyptus grandis and the recalcitrant Eucalyptus globulus were evaluated. The effect of white, blue, red and far-red radiation enrichment on microcuttings and donor plants of both species was evaluated in relation to rooting. There was no effect of radiation quality on adventitious rooting of E. grandis or when radiation treatments were applied to E. globulus microcuttings. In contrast, donor plants of E. globulus, grown in medium devoid of sucrose and exposed to far-red radiation, yielded microcuttings showing higher rooting percentage, even in the absence of exogenous auxin in the rooting medium. Sucrose in donor plant medium abolished the positive effect of far-red radiation. An increase in endogenous soluble sugars and starch contents in basal microcuttings was associated with far-red radiation treatment of donor plants. These results underline the importance of appropriate carbohydrate partitioning in donor plants for adventitious rooting of cuttings and provide a basis for understanding and overcoming rooting recalcitrance in E. globulus clones.     

Application of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> (Chlorophyceae) as supplement and/or an alternative medium for the in vitro cultivation of <Emphasis Type="Italic">Schomburgkia crispa</Emphasis> (Orchidaceae)

Schomburgkia crispa Lindley (Orchidaceae) is an epiphytic species found in gallery forests and dry vegetation in the Brazilian Cerrado. It is typically unable to germinate or exhibits low germination because of dependency on mycorrhizal associations. In vitro cultivation techniques have helped circumvent difficulties involved in propagation from seeds. Alternative media and organic biostimulant substances that reduce costs and promote satisfactory in vitro growth are constantly sought. This study evaluated in vitro multiplication and rooting of S. crispa in a modified culture medium containing extract of the microalga Chlorella sorokiniana. We analyzed supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium. The extracts were added to WPM instead of distilled water. The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively. Both growth regulators were tested at 0, 2.5, and 5.0 mg L^?1. During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L^?1 BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L^?1 IBA supplemented with microalgae extract stimulated in vitro rooting. Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone. Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa.     

Effect of growth regulators onin vitro propagation ofFicus benjamina cv. Exotica

Stem internodes with axillary buds were excised from 5-year old trees ofFicus benjamina cv. Exotica. The effect of 6-benzylaminopurine (BAP), gibberellic acid (GA₃), indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on shoot growth and proliferationin vitro was investigated. Multiple shoots were developed after 3–4 weeks from stem internodes with axillary buds incubated in Murashige and Skoog (MS) medium supplemented with phloroglucinol (PG) and BAP. Optimum shoot proliferation took place in the presence of 1.0 mg l⁻¹ BAP. Shoots obtained could be elongated in a medium with 0.5 mg l⁻¹ GA₃ prior to their rooting. The root initiation was successfully induced on MS medium either with IAA at 0.5–0.1 mg l⁻¹ or in plant growth regulator-free medium. All rooted plantlets were subsequently transferred to a peat, humus and perlite mixture in a culture room with high humidity and covered with plastic bags. After one month the plantlets were established for growing in a greenhouse. Communicated by J. TUPY     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

Abstract

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36?h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0?μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0?μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5?μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15?μM TDZ for 24?h) explants was achieved on WPM augmented with 6.0?μM TDZ and 0.5?μM NAA after 8?weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (～0.5?g) and were transferred onto WPM fortified with different concentrations (2.0–9.0?μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0?μM) and gibberellic acid (GA_3, 0.5–3.0?μM). Maximum shoot number (10.4?±?0.74) and average shoot length (4.75?±?0.71?cm) were observed in WPM enriched with 2.0?μM BAP, 1.0?μM IAA and 1.5?μM GA₃ after 8?weeks of incubation. The developed shoots (4?cm) were excised, pulse-treated for 24?h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0?μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60?days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.     

In vitro propagation of purple pitahaya (Hylocereus costaricensis [F.A.C. Weber] Britton & Rose) cv. Cebra

Limitations to large-scale propagation of purple pitahaya (Hylocereus costaricensis [F.A.C. Weber] Britton & Rose), a potential source of betalains for the food industry, can be overcome through utilization of in vitro culture technologies. In this work, successful in vitro propagation from areoles of adult purple pitahaya plants is reported. Factors affecting culture initiation, bud sprouting and growth, shoot multiplication, rooting, and acclimatization were studied. Best results for culture initiation were obtained from the central region of new joints by disinfection of large (5?C7?cm in length) explants that were subsequently divided. Explants were sequentially treated with Extran? alkaline detergent for 10?min, followed by immersion in 70?% (v/v) ethanol for 15?C30?s, a mixture of the fungicide Benomyl and the bactericide Agrymicin (2?g?l^?1 each) for 30?min, and disinfection in sodium hypochlorite (1.0?%?w/v) for 15?min. Culture of sectioned individual areoles, without removing thorns, on Murashige and Skoog medium supplemented with 15 or 30???M N ⁶-benzylaminopurine (BAP) for 3?mo with monthly subcultures, followed by transfer to the same medium with reduced BAP (0?C2???M), induced bud sprouting in over 80?% of explants, adequate growth of the shoots, with production of lateral shoots, and spontaneous rooting within 160?d. These plants were successfully acclimatized in vermiculite and peat moss (1:1), or perlite and peat moss (2:1) in the greenhouse, with over 90?% survival rate. One hundred percent of the in vitro-derived plants were successfully transferred to the field. Furthermore, these plants showed higher survival rates, larger height and increase in stem diameter than equivalent plants from the same genotype, derived from stem segments (the common clonal propagation system utilized for this species) that were simultaneously planted.     

Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)

A protocol was developed for plant regeneration of Melia azedarach L. by in vitro culture of apical meristem (0.5 mm in length). The influence of six clones was investigated. The culture procedure comprised two sequential steps: 1) Induction of shoots by in vitro culture of axillary buds from adult trees (10–15 years old) by culture on Murashige and Skoog (1962) medium (MS) supplemented with 0.5 mg·dm⁻³ BAP (6-benzylaminopurine), 0.1 mg·dm⁻³ IBA (indolebutyric acid), and 0.1 mg·dm⁻³ GA₃ (gibberellic acid). The Multiplication of the regenerated shoots was achieved in MS + 0.5 mg·dm⁻³ BAP + 0.1 mg·dm⁻³ GA₃. 2) In vitro culture of the apical meristems from the regenerated shoots in MS medium (0.7 %) supplemented with various combinations of BAP and IBA. Maximum shoot proliferation was obtained on MS medium supplemented with 0.5 mg·dm⁻³ BAP and 0.1 mg·dm⁻³ IBA. Regenerated shoots were rooted on MS + 3.5 mg·dm⁻³ IBA (4 days) followed by subculture on MS lacking growth regulators (30 days). Complete plants were transferred to soil.     

Establishment and in vitro morphogenesis of sapucaia explants (Lecythidaceae)

Lecythis pisonis Cambess, popularly known as sapucaia, has great economic and socio-environmental potential. The objective of this study was to evaluate the establishment and in vitro morphogenesis of L. pisonis under the effect of disinfecting agents, plant growth regulators, and thermal stress. The study was divided into three experiments: (i) development of the disinfection protocol by testing different concentrations and times of exposure to sodium hypochlorite (NaOCl) and different concentrations and methods of amoxicillin application, (ii) in vitro budding induction by testing different concentrations of 6-benzylaminopurine (BAP) or kinetin (KIN) supplemented to Woody Plant Medium (WPM) and Murashige and Skoog (MS) culture media, and (iii) in vitro formation from plantlets by analyzing different concentrations of indole-3-butyric acid (IBA) with different exposure times to a thermal stress of 40°C. The disinfection of stem segments was effective using 3% NaOCl and 3.0 g L⁻¹ amoxicillin solution. MS culture medium supplemented with 0.25 mg L⁻¹ BAP induced more shoots in vitro. One milligram per liter IBA promoted greater rooting in vitro, and it is not necessary for thermal stress tolerance.

     

Influence of plant growth regulators on micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br

An efficient protocol for micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br. was developed using shoot tip explants. The physiological role of cytokinin and its combination with auxins on micropropagation and in vitro flowering was investigated. The highest number of shoots (9.94 ± 0.10) and the maximum average shoot length (5.56 ± 0.35 cm) were recorded on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) (4.44 μM) and naphthaleneacetic acid (NAA) (2.69 μM). The effect of sucrose concentration on in vitro floral development was studied in plantlets cultured on MS medium supplemented with gibberellic acid (GA₃) and BAP. The highest percentage of flowering (93.2%) was obtained on MS medium supplemented with GA₃ (1.44 μM), BAP (1.33 μM) and sucrose (30 g l^?1). Root formation from the adventitious shoots was easily achieved on MS medium containing indole-3-butyric acid (IBA) (2.46 μM). The regenerated plantlets showed 86% survival rate and were phenotypically normal. The described method can be successfully employed for large-scale multiplication and in vitro flowering of T. indicum.     

High-efficiency regeneration of Agrobacterium rhizogenes-induced hairy root in apple rootstock Malus baccata (L.) Borkh

Malus baccata is widely used as a rootstock in cold regions of the world because of its cold hardiness. In this study, a highly efficient Agrobacterium rhizogenes strain 8196 transformation system was developed using in vitro-derived stem segments of M. baccata. Approximately 37?% agro-infected explants produced hairy roots when they were incubated on Murashige and Skoog (MS) medium without plant growth regulators. A total of 95?% of hairy roots exhibited glucuronidase activity. Calli were induced from putatively-transformed hairy roots, and subsequently shoots were observed within 4?weeks of culture. The influence of 6-benzyladenine (BA), indole-3-butyric acid (IBA), thidiazuron (TDZ), and gibberellic acid 3 (GA₃) on regeneration were investigated using an L₉ (3⁴) orthogonal experiment. About 73?% of shoots were regenerated when callus was incubated on MS medium along with 2.0?mg?L^?1 BA, 0.5?mg?L^?1 IBA, 0.3?mg?L^?1 GA₃, and 0.5?mg?L^?1 TDZ. Moreover, hairy root regenerants showed higher rooting ability and exhibited morphological aberrations such as shortened stem, etiolated, wrinkled and clustered leaves than those of control.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

Effects of 6-benzylaminopurine on photosystem II functionality and leaf anatomy of <Emphasis Type="Italic">in vitro</Emphasis> cultivated <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis>

Cytokinins (CKs) are often used during the in vitro cultivation of plant species. However, it is not clear how CKs, such as 6-benzylaminopurine (BAP), affect photosystem PS) II functionality and leaf anatomy over a long period of in vitro plant propagation. The aim of this study was to analyze the residual effects of BAP on the photosynthetic performance and leaf anatomy of Aechmea blanchetiana after 120 d without exposure to CKs. Aechmea blanchetiana plants previously grown in vitro were transferred to Murashige and Skoog (MS) culture media containing 0, 5, 10, 15, or 20 μM BAP. After 60 d on the MS medium with BAP, explants were subcultivated twice on the MS medium without growth regulators, first in a stationary liquid medium for 60 d and then in a solidified medium with 6 g dm^-3 agar for 60 d. Leaf anatomy, pigment content, and chlorophyll a fluorescence were assessed for plants from each treatment after 120 d on the CK-free medium. Stomatal density presented a negative linear correlation with BAP concentration. Pigment content decreased in plants subjected to previous BAP exposure. An increase in absorbed energy flux per reaction center (ABS/RC) and a sharp decrease in energy transport flux (ETo/RC) followed by an increase in energy dissipation flux (DIo/RC) also occurred. Furthermore, maximum quantum yield (F_V/F_M) decreased as a function of BAP concentration. Thus, the use of BAP during in vitro propagation of A. blanchetiana induced long-term physiological defects.     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of brown oak (<Emphasis Type="Italic">Quercus semecarpifolia</Emphasis> Sm.) from seedling explants

An efficient and reproducible method for the regeneration of multiple shoots of brown oak (Quercus semecarpifolia Sm.) has been developed in which a part of the petiolar tube containing a primary shoot is used as the explant. Explants derived from in vitro grown seedlings were cultured either on Murashige and Skoog or Woody Plant medium (WPM) containing different concentrations of benzyladenine (BAP) throughout the range of 1–20 μM. WPM supplemented with 20 μM BAP was found to be best for adventitious shoot induction and for the multiplication of individual shoots. In-vitro-produced shoots were rooted using a two-step method. Firstly, shoots were cultured on WPM containing indolebutyric acid (IBA) at either 50 or 100 μM for 24 or 48 h. Secondly, the shoots were transferred to plant-growth-regulator-free half-strength WPM. The second step not only considerably improved the rooting percentage but also minimized the formation of basal callus. The most effective first-step treatment was found to be 100 μM IBA for 24 h, which initiated rooting at a frequency of 100%. Well-rooted plants were transferred to plastic cups containing nonsterile, sieved soil and farmyard manure, hardened under greenhouse conditions, and then successfully established in pots. This procedure is suitable for use in large-scale production of plants and may have potential application in additional oak species.