Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

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.     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Effect of Auxins on Adventitious Root Development from Single Node Cuttings of Camellia sinensis (L.) Kuntze and Associated Biochemical Changes

An attempt was made to induce rooting from single node cuttings of Camellia sinensis var. TV-20 under controlled conditions and study its biochemical changes during rooting. The nodal cuttings were pretreated with different concentrations of IAA, NAA and IBA and kept in a growth chamber (25 ±2 °C, 16 h photoperiod (55 μ mol m⁻² s⁻¹) with cool, white fluorescent lamps and 65% relative humidity) for 12 h. Among the three auxins used for pretreatment, IBA showed more positive response on rooting as compared to IAA and NAA within 2 weeks of transfer to potting medium. Among four concentrations of IBA tested, 75 ppm gave maximum percentage of rooting, number of roots and root length. Therefore, IBA was used further in experiments for biochemical investigation. The adventitious rooting was obtained in three distinct phases i.e. induction (0–12 days), initiation (12–14 days) and expression (14–18 days). IAA-oxidase activity of IBA-treated cuttings increased slightly as compared to control. The activity was found to decrease during induction and initiation phases and increase during expression phase. The peroxidase activity in IBA-treated cuttings increased up to initiation phase and declined at the expression phase. Polyphenoloxidase activity increased both in IBA-treated and control cuttings during induction and initiation phase but declined slowly during expression phase. Total phenolic content was higher in IBA-treated cuttings, particularly in initiation and expression phases and it also correlated with peroxidase activity. Phenolics might be playing key role for induction of adventitious rooting, and phenolic compounds can be used as rooting enhancer in tea plant.     

Factors affecting the early gene transfer step in the development of transgenic ‘Fuji’ apple plants

This study was conducted to determine the optimal transformation conditions during early gene transfer steps necessary to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in Fuji apple plants. The use of 200 μM acetosyringone in the co-culture medium resulted in 4.7% transformation efficiency, compared with different concentrations of 0–400 μM. A 4-day co-culture period gave 1.9% transformation efficiency, compared with different co-culture periods of 1–5 days. There was no significant difference in transformation efficiency with different explant placement orientations on co-culture medium. A comparison of young leaves from plants cultured in rooting medium for 4 weeks with those cultured for 8 weeks showed that an 8-week culture period resulted in higher transformation efficiency. We therefore concluded that the efficiency of Fuji apple transformation depends on improving factors related to the gene transfer step.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.     

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.     

Influence of cytokinin levels on <Emphasis Type="Italic">in vitro</Emphasis> propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Verification of QTL linked markers for propagation traits in <Emphasis Type="Italic">Eucalyptus</Emphasis>

In a previous study, several quantitative trait loci (QTLs) affecting vegetative propagation traits were detected in a hybrid cross between Eucalyptus tereticornis and Eucalyptus globulus. The objective of this work was to confirm stable QTL linked markers (detected in different years) for propagation traits in an independent set of the same segregating population and in two related crosses involving the original E. globulus parent. Phenotypic averages of groups of individuals carrying alternative allelic forms of the stable QTL linked markers were statistically tested for significant differences. Adventitious rooting and petrification marker–trait associations, detected previously in the E. tereticornis parent, were verified in an independent sample of the original progeny. In the E. globulus parent, the QTL linked marker was only verified in one related genetic background. Verification was possible only for high-effect QTL linked markers. This study highlights the importance of sample size in QTL detection for low-heritability traits.     

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

Summary Success has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl⁻¹) + ascorbic acid (50 mgl⁻¹) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

Stimulation of root development with cyclodextrins on jojoba shoots in vitro

Summary We studied the effect of α- and β-cyclodextrins on in vitro rooting of jojoba (Simmondsia chinensis (Link) Schn.) shoots. When 0.03–0.5 mM of cyclodextrins were added to modified Murashige and Skoog culture medium containing 0.015 mM of indolebutyric acid as 1-wk pulse treatment, rooting percentage increased by approximately 100% with respect to controls. Addition of cyclodextrins induced earlier rooting. An increase in rooting following treatment with cyclodextrins was also observed in the absence of indolebutyric acid, indicating that both compounds promote rooting per se.     

In vitro propagation of two spanish endemic species of salvia throught bud proliferation

Summary Salvia valentina Vahl and Salvia blancoana Webb & Heldr subsp. mariolensis Figuerola, two endemic species of Salvia from the Mediterranean coastal region of Spain, were successfully gegenerated in vitro from adult plants using two explant types (apical and nodal segments). Maximum shoot proliferation for both species was obtained with nodal explants: for S. blancoana on Murashige and Skoog medium supplemented with 6-γ-γ-dimethylallylaminopurine at 1 mg 1⁻¹ (4.9 μM). and for S. valentina on the same medium with kinetin at 1–2 mg 1⁻¹ (4.6–9.3 μM). The influence of apical dominance, and the explant viability in culture were found to be the main differences between the two species during the shoot multiplication phase. Rooting of shoots was low, specially for S. valentina. For both species, rooting was achieved in Murashige and Skoog medium without growth regulators. In general the addition of the auxins indole 3 acetic acid or indole-3-butyric acid did not improve the rooting or, in the case of naphthaleneacetic acid, had an inhibitory effect. In the best rooting media, rooting shoots increased in length. The rooted plantlets were acclimated to ex vitro conditions and a survival percentage > 70% was obtained under greenhouse conditions. This work was carried out as an ex situ conservation method for these Spanish endemic species.     

Micropropagation of photinia employing rhizobacteria to promote root development

An alternative protocol was developed for in vitro propagation of photinia (Photinia × fraseri Dress), an ornamental shrub, using the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Azotobacter chroococcum during rhizogenesis. Shoot tips from four-year-old mature plants, cut in spring and summer, were used as initial explants. They were cultured on Murashige–Skoog (MS) medium with Gamborg’s vitamins, N⁶-benzyladenine (BA: 11.1 μM) and gibberellic acid (GA₃: 1.3 μM), obtaining 63% of established explants. The highest shoot length (22.9 mm) and multiplication rate (4.3) was achieved by cultivating for four weeks in the same basal medium supplemented with 4.4 μM BA. Both auxin induction and bacterial inoculation were used for rooting. Elongated shoots were treated with two concentrations of indole-3-butyric acid (IBA: 4.9 or 49.2 μM) during 6 days for auxin induction. Then, the shoots were transferred to an auxin-free medium and inoculated with A. brasilense Cd, Sp7 or A. chroococcum (local strain). Bacterial inoculation induced earlier rooting of photinia shoots. A. brasilense Cd with 49.2 μM IBA pulse showed a significant increase (P ≤ 0.05) in root fresh and dry weight (105%, 137%), root surface area (65%) and shoot fresh and dry weight (32%, 62%). A. brasilense Sp7 enhanced the root fresh weight (34%) and root surface area (41%) while no significant differences with A. chroococcum inoculation were detected. The PGPR inoculated micro-cuttings in combination with auxin induction pulses may play a useful role in root organogenesis of micropropagated plants.     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Micropropagation and hairy root culture of <Emphasis Type="Italic">Solanum Laciniatum</Emphasis> Ait.

Summary An efficient system for in vitro micropropagation of Solanum laciniatum Ait. has been established. Shoot induction on leaf explants was most successful on Murashige and Skoog (MS) medium supplemented with 10 μM N⁶-benzyladenine (BA) and 1 μM α-naphthaleneacetic acid (NAA). BA (13 μM) was optimal for further shoot multiplication, and rooting of separated shoots was achieved on medium without plant growth regulators. At each subculture, 20–25 shoots were obtained on each explant, from which six to eight were suitable for separation and further rooting. Leaf explants grown in vitro were successfully infected by Agrobacterium rhizogenes ATCC 15834. The established hairy root culture was, on the basis of dry weight, more productive when grown on half-strength MS medium than on full-strength MS (3% sucrose) and full-strength MS (6% sucrose) medium. The amount of solasodine-containing glycoalkaloids in hairy roots as measured by a colorimetric method was 0.3–1% of dry weight, which is higher than in the shoot culture (0.5% of dry weight) and lower than in leaves of in vivo-grown plants (1.1–1.4% of dry weight). The amount of solasodine-containing glycoalkaloids in leaves of in vivo-grown plants of S. laciniatum was similar to the related species Solanum aviculare Forst. Both species are morphologically similar, therefore we effectively distinguished them by flow cytometry. The genome size of S. laciniatum was determined as 4.03 pg and the genome size of S. aviculare as 1.69 pg.     

<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.     

TDZ-specific plant regeneration in salad burnet

Various explants of salad burnet (Poterium sanguisorba L.=Sanguisorba minor Scop.) were cultured on semi-solid MS or B5 media containing factorial combinations of various plant growth regulators. Hypocotyl and petiole explants, after initial callus stage, regenerated prolific adventitious shoots via organogenesis, when placed on Murashige and Skoog basal medium containing 1–2 μM α-naphthaleneacetic acid and 4-20 μM thidiazuron. Any other growth regulator combination tested failed to respond. The addition of the biocide, Plant Preservative Mixture, was effective in controlling contamination and did not impair regeneration. Elongated shoots at 2–4 cm were transferred to rooting medium containing semi-solid Murashige and Skoog plus 1 μM α-naphthaleneacetic and rooted plants were transferred to the glasshouse. This is the first report on in vitro plant regeneration within the genusPoterium. This revised version was published online in June 2006 with corrections to the Cover Date.