Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

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.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Tylophora indica (Burm. f.) Merrill, via enhanced axillary bud proliferation from nodal explants collected from young shoots of a two-year-old plant. The physiological effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)], ascorbic acid (AA), different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.6 ± 0.71) of shoots and the maximum average shoot length (5.2 ± 0.31 cm) were recorded on MS medium supplemented with 2.5 μM BA, 0.5 μM NAA and 100 mg/l AA at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with 0.5 μM IBA. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with a 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long-term in vitro conservation of T. indica.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

High-efficiency plant regeneration from leaf explants of male himalayan poplar (Populus ciliata wall.)

Summary This study reports a protocol for high-efficiency plant regeneration from leaf explants of male Himalayan poplar (Populus ciliata Wall.). Shoots were regenerated at high frequencies from explants grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ kinetin and 0.2 mg l⁻¹ indole-3-acetic acid (IAA). Regenerated shoots developed roots in MS medium supplemented with 0.1 mg l⁻¹ IAA. Himalayan poplar plantlets could be produced within 2 mo. after acclimatization in a sterile mixture of sand and soil.     

Genotypic and media effects on plant regeneration from cotyledon explant cultures of someBrassica species

Conditions were established for efficient plant regeneration from cotyledon explant calli in different cultivars ofBrassica juncea, B. campestris andB. carinata on Murashige & Skoog's (MS) medium supplemented with various combinations of cytokinins and auxins. Regeneration frequency, however, varied with genotype and the different growth hormone combinations in media. Almost in all species, MS medium with zeatin (1.0 mg 1^-1) and IAA (0.1 mg l^-1) was found to be best for shoot organogenesis followed by the ones containing high kinetin (2.0 mg l^-1) and low IAA (0.02 or 0.2 mg l^-1) concentrations. On these media, the cotyledonary explants invariably underwent callusing followed by multiple shoot formation, which could be separated and subcultured for further propagation. Number of shoots per cotyledon explant cultured varied from 0 to as many as 50. InB. juncea andB. campestris, the regeneration frequency declined sharply in the absence of auxin in medium. BAP in combination with NAA yielded no or a reduced number of shoots. Shoot organogenesis also declined with the reduction in photoperiod from continuous light to 16 hours. Shoots were easily rooted during prolonged incubation on the same medium and whole plants were transferred to pots in the greenhouse and grown to maturity.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - MS medium after Murashige & Skoog [8] - NAA -napthaleneacetic acid - ZEA Zeatin     

High-frequency regeneration and transformation ofRaphanus savus

We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s (MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing 0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

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     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

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     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997