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.     

In vitro propagation of Fibigia triquetra (DC.) Boiss., a rare stenoendemic species

A rapid clonal propagation method for Fibigia triquetra (DC.) Boiss. (Brassicaceae), a rare Croatian stenoendemic species, has been developed. Shoots originated from aseptically germinated seeds were used for culture initiation. The highest multiplication rate of 9.2 shoots per explant was achieved in a 4-weeks-culture period in the third subculture on half strength Murashige and Skoog medium supplemented with 0.5 μM 6-benzylaminopurine and 2.9 μM gibberellic acid. Excised shoots were rooted best with addition of 8.6 μM indole-3-butyric acid on the same basal medium. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct plant regeneration from different explants through micropropagation and determination of secondary metabolites in the critically endangered endemic Rhaponticoides mykalea

Direct plant regeneration from different explants, micropropagation and determination of secondary metabolites were studied in the critically endangered endemic Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab & Greuter. Seed germination was achieved by damaging the seed coat and cultivating the embryos on Woody Plant Medium (WPM), of which 40% germinated. The epicotyls and cotyledonary petioles of seedlings were used as initial explants and direct shoot regeneration was obtained on WPM containing 2.22 μM 6-benzyladenine (BA). WPM medium supplemented with 2.22 μM BA and 4.92 μM indole-3-butyric acid (IBA) significantly improved the production of multiple shoots, resulting in an average of 5.6 shoots per explants. The highest rooting of shoots (35.6%) was observed with WPM medium containing 19.68 μM IBA with 990 μM putrescine. Plantlets with well-developed roots were transferred to soil and acclimatised within a plant growth chamber. Acclimatised plants showed 100% survival rate and remained healthy. As a part of our study, the content of secondary metabolites in three tissue culture regenerated lines were determined by HPLC analysis. Chlorogenic acid, Quercetin and scutellarin were confirmed secondary metabolites of R. mykalea.     

Micropropagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L^?1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L^?1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

<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.     

Multiple shoot formation from cotyledonary node segments of Eastern redbud

A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 μM BA and 0.5 or 1.0 μM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 μM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.     

Regeneration of Lonicera tatarica plants via adventitious organogenesis from cultured stem explants

We describe an efficient process for the regeneration of Lonicera tatarica plants from cultured stem sections. Induction of multiple shoots was achieved directly from cultured stem cuttings. The highest regeneration rate was achieved on Gamborg's B5 medium supplemented with 4% sucrose and 0.8% Difco bacto-agar in the absence of hormones. Differentiated shoots were elongated for 5-7 days on induction medium supplemented with 0.5 mg/l GA₃. Shoot induction and elongation experiments were carried out using original stem explants from either 2-, 6-, or 18-month-old donor plants. The age of the donor plant had no noticeable effect on either process. However, rooting of elongated shoots occurred only with shoots derived from 2-month-old donor plants. Rooting efficiency and proliferation were highest on half-strength WPM medium supplemented with 2 µM indole-3-butyric acid and 0.6% Keylis agar. The plants regenerated from stem explants were morphologically normal, and levels of loganin and secologanin were comparable to those detected in plants grown from seed and maintained through vegetative propagation.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

珍稀濒危植物珙桐胚的萌发与快速繁殖

首次采用均匀设计和回归分析方法对影响珙桐（Davidia involucrata）胚萌发的3种因素（基本培养基、赤霉素和活性炭）进行优化,并利用萌发胚建立了珙桐的高效再生体系。利用回归方程得出胚萌发的最优条件为：在含有0．5mg·L^-1 IBA和1mg·L^-16·BA的1/2MS培养基中添加2．85mg·L^-1GA3和0．25g·L^-1活性炭。在验证实验中珙桐胚萌发率为87．72％,与预测值无显著差异。最优的芽诱导与增殖条件为：添加0．1mg·L^-1NAA和2．0mg·L^-16-BA的WPM培养基,增殖系数为4．34。在生根培养基中增殖的芽生根率高达90％,移栽成活率为71％。     

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, ‘Malas Saveh’ and ‘Yousef Khani’. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for ‘Malas Saveh’ and 9.2 μM for ‘Yousef Khani’, resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.     

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.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

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.     

Rapid plant regeneration,validation of genetic integrity by ISSR markers and conservation of Reseda pentagyna an endemic plant growing in Saudi Arabia

Reseda pentagyna is the only endemic species among the seven species of the genera Reseda found in Saudi Arabia. Probably no information is available on regeneration by conventional method of regeneration through seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerate and multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after four weeks, when shoot cuttings cultured on MS containing BA at 1.0 µM. Other cytokinins e.g., Kn, 2iP and TDZ found to be less effective in bud induction and shoot multiplication. Individual shoots were rooted on MS medium supplemented with various auxins at 0.5–5.0 µM concentrations. The IBA (1.5 µM) supplemented MS media induced maximum (83.3%) rooting. The plantlets were acclimatized and hardened under greenhouse conditions in plastic pots containing soil and farm yard manure with 95.0% success. The protocol developed would help to multiply the plant as well as conserve them in natural habitat. This can also be utilized to obtain active constituents for pharmaceutics and genetic manipulations.     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.     

Rapid multiplication of Jasminum officinale L. by in vitro culture of nodal explants

Jasminum officinale L. (Fam. - Oleaceae) a shrub, noted specially for its aroma, has been micropropagated successfully by culturing nodal segments. MS basal media with 3% sucrose and supplemented with 6-benzyladenine, at 17.76 μM and 1-naphthaleneacetic acid at 0.53 μM concentration was used for shoot proliferation. For elongation, BA and kinetin, individually and also in combination with NAA were supplemented in the medium. 4 to 5 cm long shoots were transferred to liquid rooting medium. Rooted plants grew normally under natural conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [<Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.     

An efficient method for regeneration of lavandin (Lavandula x intermedia cv. 'Grosso')

An efficient protocol for the regeneration of lavandin (Lavandula x intermedia cv. 'Grosso') is reported. Thiadazuron (9 μM), a plant growth-modulating phenylurea, was used to induce callus formation and shoot initiation from cultured leaf explants. Newly emerged shoots were maintained on media containing 0.05 μM naphthaleneacetic acid to allow maturation, and then transferred to media containing 2.9 μM indole-3-acetic acid to allow root formation. The phenolic control agents polyvinylpyrrolidone (PVP), ascorbic acid, 2-aminoindane-2-phosphonic acid, and activated charcoal were tested for their ability to prevent shoot browning and death in culture. All agents except PVP were found to be effective, with ascorbic acid being most consistent in promoting development of healthy mature shoots. The effect of light type (red light vs. white light) and culture medium composition (full- and half-strength Murashige and Skoog or Llyod and McCown’s woody plant medium (WPM)) on rooting efficiency was also evaluated. Cultures on half-strength WPM in white light were found to have the highest rooting efficiency. Additionally, application of the polyamines putrescine, spermine, and spermidine were tested for their effect on rooting. While rooting efficiency was not improved with any of the treatments, spermine and spermidine were found to have an inhibitory effect at concentrations greater than 10 μM.