In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

In vitro propagation of Coleus forskohlii Briq., a threatened medicinal plant

In vitro clonai multip1ication of Coleus forskahlii Briq a threatened plant, has been achieved on MS medium supplemented with Kn (2.0 mg/l) and IAA (1.0 mg/l) using nodal segments as explants, Shoots multiplied at a rate of 12 — fold every six weeks. Rooting was achieved upon transfer of shoots onto MS medium containing IAA (1.0 mg/l). The micropropagated plants were successfully established under field conditions. Forskolin content in tubers of plants obtained by micropropagation was found to be 0.1%, the same as that found in wild plants. This micropropagation procedure should be useful for conservation as well as production of this important plantAbbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) basal medium - NAA -naphthalene acetic acid     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962     

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 M) singly or in combination with -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Successful micropropagation protocol of <Emphasis Type="Italic">Piper methysticum</Emphasis>

An efficient in vitro propagation of kava (Piper methysticum) was established. Utilizing 15-d-old tender shoots from dormant auxiliary buds as explants, significant induction of vigorous aseptic cluster shoots was achieved in Murashige and Skoog (MS) medium containing 0.5 mg dm⁻³ 6-benzyladenine (BA), 0.5 mg dm⁻³ indole-3-acetic acid (IAA), and antibiotics after 30 d. In vitro rooting was achieved at 100 % efficiency in MS medium containing 0.75 to 1.00 mg dm⁻³ IAA or indole-3-butyric acid and 3 % sucrose. The most robust and long roots were observed in medium with IBA. Moreover, the embryonic callus was induced from petioles in MS medium supplemented with 1.0 mg dm⁻³ BA and 0.1 mg dm⁻³ IAA, of which 70 % differentiated into shoots in the presence of 1.0 mg dm⁻³ BA and 0.5 mg dm⁻³ IAA.     

In vitro plant regeneration from cotyledon and hypocotyl segments in two bell pepper cultivars

In vitro plant regeneration has been obtained from Capsicum annuum cvs. Pico and Piquillo. Shootbuds were induced from hypocotyl and cotyledon segments after 15–20 days of culture on MS basal medium supplemented with IAA and BAP or Zeatin. Shoot-buds grew into rosettes that rooted in MS plus NAA (0.1 mg/l) and IBA (0.05 mg/l) after 15 days. The small plantlets were successfully transferred to pots with a mixture of peat and perlite and maintained under greenhouse conditions. Elongation took place when the plantlets were growing in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA alpha-naphthalenacetic acid - Z Zeatin     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

In vitro establishment of a cycloclonal chain from nodal segments and apical buds of adult hazel (Corylus avellana L.)

Apical buds (0.5 cm) and nodal shoot segments (1.5 cm) excised from: A) field-grown branches, B) newly developed shoots from the forced outgrowth of axillary buds on A branches, C) newly developed shoots from the forced outgrowth of axillary buds on A branches submitted to cold storage were used as primary explants. Results indicate that three months cold storage greatly increases morphogenic capacity and reduces contamination and oxidation of tissues. Consequently, a multiplying chain could be easily established by culturing the tissues on a modified Murashige & Skoog (1962) medium plus 6-benzyl-aminopurine 5 mg l^-1, indole-3-acetic acid 0.01 mg l^-1 and gibberellic acid 0.1 mg l^-1. During the initiation and proliferation phases, both the proliferation and the elongation rate were significantly increased when a double-phase culture system (Viseur 1987) was used, giving rise to a higher microplant production than the one obtained using previously described methods. Plant regeneration was achieved by immersing the single microshoot's basal end in an IBA (0.1–1 mg ml^-1) solution for 10 s followed by a 20-day culture on a 1/2 MS2 medium.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - MS1 and MS2 modified Murashige & Skoog media - NAA 2-maphtaleneacetic acid     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid