<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot regeneration of the medicinal plant meadowsweet (<Emphasis Type="Italic">Filipendula ulmaria</Emphasis> (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA₃), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA₃. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.     

Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N ⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.     

Micropropagation and slow growth conservation of cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton)

Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about 70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of 20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation was detected among the in vitro-conserved plants compared with the mother plants.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

Plantlet regeneration from leaf derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.     

Plant regeneration from embryogenic cells-derived protoplasts of Bupleurum falcatum

Hypocotyl segments of Bupleurum falcatum L. formed embryogenic calluses when cultured on Murashige and Skoog's (MS) medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures were initiated by placing calluses into medium with 0.45 μM 2,4-D. Protoplasts were enzymatically isolated from suspension cultures. They were plated at a density of 5 × 104 protoplasts per ml on MS medium supplemented with 9% mannitol, 9.0 μM 2,4-D, 4.4 μM BA, 4.6 μM kinetin, and 0.6% Seaplaque agarose. After four weeks of culture, microcalluses were formed and subsequently transferred to MS solid medium with 18.1 μM 2,4-D. Upon transfer to MS basal medium, microcalluses gave rise to somatic embryos at a frequency of approximately 10%. They subsequently developed into plantlets. The regenerants were successfully transplanted to potting soil and grown to maturity in a greenhouse. The regenerants had the normal chromosome number of 2n=2x=20 and did not show morphological aberrancy. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.     

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS⁺ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR⁺ plants.     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.