Micropropagation of a medicinal plant, Plantago major L.

An efficient micropropagation protocol was developed for an important medicinal plant, Plantago major L. For this purpose, it is recommended to culture shoot-tips on modified MS medium [412.5 mg dm-3 NH4NO3 and 340 mg dm-3 KH2PO4] supplemented with 50 g dm-3 glucose and 0.5 μM 6-benzylaminopurine. Maximum rooting frequency was obtained at 1 μM naphthaleneacetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation and essential oil characterization of Plectranthus amboinicus (Lour.) Sprengel,an aromatic medicinal plant

Plectranthus amboinicus (Loureiro) Sprengel is a therapeutically valued herb highly sought by various industries. This has led to its uncontrolled harvest from the wild leading to the risk of extinction. As an alternative, commercial propagation of P. amboinicus is desirable to avoid further depletion of wild populations. This paper describes the establishment of an efficient protocol for the multiplication, rooting and acclimatization of P. amboinicus. The greatest frequency of shoot multiplication of P. amboinicus was on semi-solid Murashige and Skoog (MS) medium supplemented with 0.4 mg L⁻¹ 6-benzylaminopurine (BAP). Media supplemented with higher concentrations of BAP or used in combination with gibberellic acid (GA₃) produced abnormal plantlets. Optimal rooting was observed on plantlets cultured in half strength MS medium. Acclimatization was achieved by transferring the rooted plants onto sterile peat-moss moistened with MS medium and exposed to the glasshouse environment gradually over a period of 2 months. P. amboinicus responded readily to tissue culture, but careful attention was required to media formulation to avoid abnormal plantlet development. The composition of essential oils extracted from field grown plants and in vitro microshoots were comparable.

     

Micropropagation of Cunila galioides,a popular medicinal plant of south Brazil

Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.     

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Summary Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success. Part of the PhD thesis of P. R. Malla.     

Micropropagation of the medicinal plant, Scilla natalensis Planch.

Primary bulb explants of Scilla natalensis were cultured in vitro on modified MS medium. Some of these explants initiated shoots, which provided a sterile source of secondary leaf and bulb explants. The secondary explants responded similarly to various combinations of plant growth regulators. Shoots were initiated spontaneously on medium containing no plant growth regulators. The number of shoots initiated was increased by the addition of kinetin or thidiazuron (TDZ) alone, but was reduced by the addition of indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) alone. Optimal shoot initiation occurred on medium containing 1 to 2 mg l^–1 kinetin and 1 to 2 mg l^–1 IAA. These shoots were rooted on medium containing 1 mg l^–1 IAA. The plantlets were successfully acclimatised in the misthouse/shadehouse.     

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Summary Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N⁶-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N⁶-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N⁶-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N⁶-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.     

Micropropagation of Ajuga bracteosa, a medicinal herb

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25?±?2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.     

Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high-frequency axillary shoot proliferation

A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal segments from mature plants. Of the three different cytokinins – N⁶-benzyladenine (BA), kinetin, and thidiazuron – evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot formation was a function of cytokinin activity alone, faster bud break coupled with an enhanced frequency of shoot development (92%) and internode elongation were dependent on the synergistic influence of gibberellic acid (GA₃) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly influenced by the explanting season. By repeated subculturing of nodal segments harvested from the in vitro-formed axenic shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA₃, prolific shoot cultures free from proximal callusing and showing a high-frequency multiplication rate were established. The percentage shoot multiplication (98–100%) as well as the number of shoots per node (six to eight) were highest during the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%) in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic acid and indole-3-butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transfer. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics as well as vegetative and floral morphology. Received: 10 January 1998 / Revision received: 17 June 1998 / Accepted: 8 July 1998     

Micropropagation protocol for Antigonon leptopus an important ornamental and medicinal plant

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3?min followed by MC 0.1% for 2?min while, using each of them individually (Clorox 20% or MC 0.1%) for 5?min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2?mg/l. The highest numbers of shootlets/explant were obtained when 2.0?mg/l of BAP or 0.5?mg/l BA?+?0.2?mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0?mg/l each combined with 0.2?mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36?bp, micropropagated plants showed 95.2% similarity in relation to mother plant.     

Micropropagation and encapsulation of medicinally important Chonemorpha grandiflora

Summary This study describes a protocol for in vitro propagation of Chonemorpha grandiflora (Roth) S.M. & M.R. Almeida (Apocynaceae) through axillary bud proliferation and rooting. For shoot induction, the combination of 13.3 μM N ⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) in Murashige and Skoog (MS) medium was better than other growth regulators. Liquid medium was superior over solid medium for root formation and growth. IBA was more effective than other auxins for root induction. Addition of silver nitrate in the presence of IBA significantly increased the number, length, and branching of roots. Shoot tips encapsulated in medium containing 0.49 μM IBA and 11.7 μM silver nitrate exhibited 95% conversion to plantlets. This protocol enables the production of 250 plantlets from a single nodal explant within 7 mo. Plantlets showed 90% survival after acclimatization in soil and were morphologically similar to the source plant under field conditions.     

Micropropagation and assessment of genetic stability in Celastrus paniculatus: An endangered medicinal plant

A highly efficient protocol for in vitro regeneration of an indigenous, endangered medicinal plant Celastrus paniculatus was achieved using nodal explants. Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L naphthaleneacetic acid (NAA) showed maximum percentage of shoot multiplication (83.4%) with 8.2 shoots/explants. Maximum rooting of 73.3% with 4.8 roots/shoot was achieved on half-strength MS media supplemented with 0.5 mg/L indole-3-acetic acid (IAA) and the percentage of survival was 91% after acclimatization. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker study confirmed genetic stability for in vitro raised explants by showing 100% monomorphism. High multiplication rate associated with genetic stability ensure the efficacy of the present in vitro clonal propagation protocol of this important medicinal plant species.     

Agrobacterium rhizogenes mediated transformation of the medicinal plant Decalepis arayalpathra and production of 2-hydroxy-4-methoxy benzaldehyde

Agrobacterium rhizogenes mediated transformation of Decalepis arayalpathra, an ethnomedicinal plant, was achieved by infecting juvenile hypocotyl explants with different strains, including A4, MTCC 532, TR105 and LBA 5402. Hypocotyl explants induced hairy roots at a higher frequency (53.2 ± 0.3 %) than cotyledons (32.1 ± 0.2 %) when infected with the most virulent strain TR105. The explants co-cultivated 48 h in half-strength salts and vitamins of Murashige and Skoog basal medium (half-MSB) induced hairy roots either directly from the wounds or followed by the formation of gall like structures. Irrespective of the explants, the strain MTCC 532 induced callus alone. The root initials on the galls proliferated vigorously and elongated more rapidly when they were segmented and subcultured on half-MSB medium than the proliferation and elongation of directly emerged roots. The established hairy roots showed intermittent gall formation which was the active sites for hairy roots induction. The molecular evidence of rol A and rol C gene integration was confirmed by PCR amplification and southern blot hybridization. Growth of the hairy roots was undertaken by measuring root growth unit after culturing root tips in half-MSB solid medium and determined fresh weight/dry weight/conductivity during time-course study in shake flask cultures. The maximum biomass and accumulation of the root specific compound, 2-hydroxy-4-methoxy benzaldehyde (MBALD) (0.22 % dry weight), was recorded at the 6th week of growth, which was more than that observed in normal root cultures (0.16 % dry weight).     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Distribution,status and conservation of Lilium polyphyllum (Liliaceae), a critically endangered medicinal plant from India

Field survey conducted to understand habitat, distribution, population structure and conservation status of Lilium polyphyllum. Three populations (total 649 individuals) are in decline because of habitat degradation, agriculture invasion and over exploitation. Our finding confirmed critically endangered status of the species, although with new criteria. We recommended integrated conservation plan.     

Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn.

An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl₂·2H₂O). A gelling matrix of 4?% Na-alginate and 100?mM CaCl₂·2H₂O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00?±?2.09?%) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0???M 6-benzyladenine (BA), 0.5???M indole-3-acetic acid (IAA) and 30.0???M adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5???M ??-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.     

Micropropagation,antioxidant properties and phytochemical assessment of Swertia corymbosa (Griseb.) Wight ex C. B. Clarke: a medicinal plant

Swertia corymbosa (Griseb.) Wight ex C. B. Clarke, a valuable medicinal plant, has been investigated for its regeneration potential using nodal explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyl adenine (2iP), thidiazuron (TDZ), and zeatin (Z)] used as supplements with MS, BA at 4.40 μM concentration proved best for multiple shoot induction yielding 26.50 ± 0.26 shoots after 12 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin BA (4.40 μM) favoured shoot multiplication. A mean number of 35.78 ± 0.81 shoots were produced per explant. Additive effect of BA (4.40 μM) in combination with Kn (4.64 μM) produced highest number of shoots (83.20 ± 4.29). Addition of GA₃ (1.4 μM) to the above medium not only favored shoot elongation but also enhanced the number of shoots (113.98 ± 3.80). The microshoots were rooted successfully on half-strength MS medium supplemented with 9.8 μM of IBA. The plantlets were successfully transferred to hardening medium containing vermiculite with 87 % survival rate. Screening of the antibacterial, antioxidant activity and estimation of total phenolic and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolic and flavonoid content than in vitro propagated plants. The content of secondary metabolites in wild-grown plants and in vitro propagated plants was determined by HPLC coupled with ESI-MS and the presence of loganic acid, swertiamarin, sweroside, gentiopicroside, isovitexin, amoroswertin, amarogentin, gentiacaulein, decussatin, and swertianin in the samples were confirmed. Gentiopicroside (40.726 mg/g) and swertianin (29.598 mg/g) were found to be the major compounds which may be responsible for the antimicrobial and antioxidant activities. The results of the present study confirmed the therapeutic potency of S. corymbosa used in the traditional medicine; in addition, the protocol for in vitro production developed in the present study could be applied for mass multiplication and for the conservation of germplasm.     

Micropropagation of Centella asiatica (L.), a valuable medicinal herb

A protocol is described for rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Centella asiatica (L.) by enhanced axillary bud proliferation in nodal segments isolated from mature plants. Although bud break was dependent on BA supply, the synergistic combination of 22.2 μM BA and 2.68 μM NAA induced the optimum frequency (91%) of shoot formation as well as shoot number (4 to 5 shoots per node). Subculturing of nodal segments harvested from the in vitro derived axenic shoots on the multiplication medium enabled continuous production of healthy shoots with similar frequency. MS medium supplemented with 6.7 μM BA and 2.88 μM IAA was found most suitable for shoot elongation. Rooting was highest (90%) on full-strength MS medium containing 2.46 μM IBA. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics. This micropropagation procedure could be useful for raising a stock of genetically homogenous plant material for field cultivation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation of Croton sublyratus Kurz – a tropical tree of medicinal importance

A shoot tip culture procedure has been developed for the rapid multiplication ofCroton sublyratus Kurz, a tropical plant species cultivated in Thailand for the production of an anti-ulcer medicine, plaunotol. Optimum conditions were established : (1) for the regeneration of shoots from shoot tips: (2) for axillary shoot formation and rooting and (3) for adaptation of regenerated plants to the open ground. The results demonstrate the feasibility of applying the shoot tip culture technique for enhancing production of plaunotol by cultivating uniform populations ofC. sublyratus with higher plaunotol levels.