Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

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.     

Regeneration of Syngonium podophyllum ‘Variegatum’ through direct somatic embryogenesis

A simple and effective method of regenerating Syngonium podophyllum ‘Variegatum’ via direct somatic embryogenesis has been established. Leaf and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with either α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos directly formed at one or two sides of petiole explants on MS medium supplemented 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA or 2.0 mg l⁻¹ TDZ with 0.2 mg l⁻¹ NAA or with 0.2 and 0.5 mg l⁻¹ 2,4-D, respectively. The frequency of petiole explants with somatic embryos produced was as high as 86% when cultured on medium containing 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA. Up to 85% of somatic embryos were able to germinate after transferring onto medium containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ NAA. Approximately 50–150 plantlets were regenerated from a single petiole explant. However, there was no somatic embryo formation from leaf explants regardless of growth regulator combinations used. Regenerated plantlets from petiole explants were stable and grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber)

 The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3–6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 μm 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μm 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10⁻⁴ m kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots. Received: 19 April 1999 / Revision received: 11 August 1999 / Accepted: 17 August 1999     

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.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Cultural behavior of protoplasts from different organs of eggplant (solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 Em^-2s^-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl^-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl^-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×10³ protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×10³ and 1,220.4×10³ protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl^-1) + zeatin (0.5 mgl^-1) + NAA (1 mgl^-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl^-1) + IAA (0.2 mgl^-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl^-1) + NAA (0.5 mgl^-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.Abbreviations (IAA) Indol-3-acetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (NAA) naphthale-neacetic - (BAP) 6-benzylaminopurine - (MS) Murashige and Skoog basal medium - (CPW) Cell and Protoplast Washing solution     

Continuous callus production and regeneration of garlic (Allium sativum L.) using root segments from shoot tip-derived plantlets

Root segments from shoot tip-derived plantlets of the garlic (Allium sativum L.) clones `DDR7099', `PI383819', and `Piacenza' were utilized as an explant source for continuous, friable callus production. The best callus production occurred on root segments initially cultured on medium with 4,5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 weeks, then subcultured to medium with 4.7 μm 4-amino-3,5,6-trichloropicolinic acid (picloram) +0.49 μm 6-(γ-γ-dimethylallylamino)purine (2iP) for 8 weeks. Embryogenic, friable callus was transferred to liquid medium for 1 month and then transferred to solid regeneration medium for 14 weeks. The best shoot and root regeneration (85.3% and 35.8%, respectively) occurred on 4-month-old calli from the clone `DDR7099'. In all clones, regeneration rate decreased as callus age increased. Received: 14 October 1997 / Revision received: 26 December 1997 / Accepted: 12 January 1998     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Micropropagation of gum karaya (Sterculia urens) by adventitious shoot formation and somatic embryogenesis

Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 μm N⁶-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 μm indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 μm 2,4-dichlorophenoxyacetic acid and 8.90 μm BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 μm thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field. Received: 26 November 1997 / Revision received: 14 April 1998 / Accepted: 11 May 1998     

Callus formation and plant regeneration from tubercles of Coryphantha elephantidens (Lem.) Lem.

Summary Callus cultures were established from pith tissue of Coryphantha elephantidens (Lem.) Lem. on Murashige and Skoog (MS) basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin. Highest shoot regeneration frequency was observed on a medium containing 6.9 μM kinetin and 2.3 μM 2,4-D under 30 μE m⁻² s⁻¹ light intensity with a 16-h photoperiod. Calluses retained organogenic potential throughout several passages of subculture (18 mo.). Shoots were rooted on MS medium without plant growth regulators. All (100%) plantlets transplanted to soil survived acclimatization. Regenerated plants showed good overall growth and were morphologically similar to the mother plants.     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

Efficient plant regeneration from protoplasts of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis> via organogenesis

A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 × 10⁵ protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 × 10⁵ protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 μM 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 μM 1-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5–7 days. When cultured in a liquid medium with 5.4 μM NAA and 8.9 μM BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 μM indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 × 10⁵ protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μm benzyladenine (BA)+0.54 μm naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N⁶-(Δ²-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μm kinetin+0.54 μm NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction. Received: 20 August 1997 / Revision received: 29 December 1997 / Accepted: 5 February 1998     

Efficacy of different growth regulators at different stages of somatic embryogenesis <Emphasis Type="Italic">Eryngium foetidum</Emphasis>L.—A rare medicinal plant

Summary High-frequency somatic embryogenesis and plant regeneration were achieved on callus developed from root, stem disc, leaf, and scape explants of Eryngium foetidum L. (Apiaceae). Calluses developed on Murashige and Skoog (MS) medium fortified with 5.37–10.74 μM α-naphthaleneacetic acid (NAA) in combination with 2.32 or 4.65 μM kinetin (Kn) formed somatic embryos after transfer to liquid half-strength MS (1/2MS) mediumwith 2.69 μM NAA and 1.16 μM Kn. Furthermore, promotion of embryo formation was also observed in callus developed on medium with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and Kn after transferral to 1/2MS liquid medium containing 10% coconut water (CW)/coconut toddy (CT). Somatic embryos developed into plantlets at the highest frequency (98%) after transferral to solid 1/2MS medium containing 10% CW/CT. All the plantlets were acclimatized in soil and survived under field conditions, and they were morphologically indistinguishable from the source plant.     

Induction of somatic embryos from cultures of <Emphasis Type="Italic">Agave vera-cruz</Emphasis> Mill.

Somatic embryogenesis from cultures of shoot apices, cotyledon and young leaves of in vitro shoots of Agave vera-cruz Mill. was studied. Embryogenic callus was obtained when explants were cultured on Murashige and Skoog’s (MS) medium (1962) supplemented with L₂ vitamins, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-d) or 5.37 μM ∝-naphthalene acetic acid (NAA). Somatic embryos differentiated from this embryogenic callus upon subculture to maturation/conversion medium containing cytokinin either alone or with auxin and l-glutamine. The best combination of growth regulators for development of somatic embryos was found to be 5.37 μM naphthalene acetic acid plus 0.91 μM zeatin and 40 g/l sucrose. The conversion frequency of somatic embryos to plantlets varied from 46–50%. Rooted plantlets were transferred directly to pots containing a soil, sand, and manure mixture without any hardening phase with 96–98% survival of the plantlets. Based on the histological observations, the potential origin of the somatic embryo is discussed.