An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Effects of plant growth regulators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, nirgundi (Vitex negundo L.)

Summary A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Rapid in vitro propagation of Saussurea lappa, an endangered medicinal plant, through multiple shoot cultures

Summary Rapid micropropagation of Saussurea lappa C. B. Clarke, an endangered medicinal plant endemic in the valley of Kashmir and western Himalayas of northern India, was achieved by culturing shoot tips (0.5–1 cm) of 2-wk-old seedlings on Murashige and Skoog’s medium (MS) supplemented with thidiazuron (TDZ, 0.45 μM). Although callus-free multiple shoots were obtained both on N⁶-benzyladenine- (BA) and TDZ-containing media, TDZ was most effective (90%) in inducing multiple shoots. Shoot tips containing proliferative buds were divided into equal halves and subcultured on MS liquid medium containing 0.225 μM TDZ for further multiplication and elongation. Multiplication of induced shoot buds was more effective when cultured in liquid medium than on agar-solidified medium. Shoots (8–10 cm) developed were rooted in MS medium containing naphthaleneacetic acid (NAA, 1.07 μM). Micropropagated plantlets were successfully transferred to soil after hardening.     

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L.

A protocol for multiple shoot bud induction and plant regeneration from leaf segment-derived callus of Ruta graveolens has been developed. Maximum organogenic callus induction frequency (70.6 ± 2.33%) was observed on Murashige and Skoog (MS) medium supplemented with 10 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus when transferred to shoot induction media (MS nutrients supplemented with 6-benzyladenine (BA), kinetin (Kn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) in various concentrations and combinations). The highest shoot multiplication (92.3%) was observed on MS medium with 7.5 µM BA and 1.0 µM NAA. Regenerated shoots were rooted in vitro on MS containing 0.5 µM IBA. Plantlets with well developed root and shoot systems were successfully acclimated (90%) and established in earthen pots containing garden soil; they exhibited normal morphology and growth characteristics.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Tissue culture response of Beta germplasm: callus induction and plant regeneration

Callus cultures of 18 sugarbeet (Beta vulgaris) lines, two accessions of B. maritima and a B. macrocarpa accession were initiated from aseptically germinated seeds. Plant regeneration through organogenesis was obtained either on MS or B5 medium containing various concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 2,3,5-triiodobenzoic acid (TIBA) and abscisic acid (ABA). Genotypes differed in their abilities of callus formation and regeneration: seven out of 18 sugarbeet lines, and an accession of B. maritima were capable of regenerating plantlets. Our data also indicated that 2 M TIBA promoted morphogenesis from callus culture in the presence of 5 M BAP.     

Indirect organogenesis and plant regeneration in Helicteres isora L., an important medicinal plant

Abstract    Helicteres isora is a medicinal plant effective against asthma, diabetes, hypolipidemia, HIV, polio besides a good source of diosgenin. Seed dormancy and low natural fruit production rate make this plant a perfect candidate for developing an in vitro regeneration method. However, to date, no such work has been procured in this plant. An efficient method for plant regeneration via shoot organogenesis from callus cultures has been developed using nodal explants in H. isora. Murashige and Skoog (MS) media counting 2,4-Dichlorophenoxyacetic acid (2,4-D, 2.26 to 13.57 μM), Indole-3-acetic acid (IAA, 2.85 to 17.13 μM), Indole-3-butyric acid (IBA, 2.46 to 14.70 μM), 6-Benzylaminopurine (BA, 2.22 to 13.32 μM) and Kinetin (Kin, 2.32 to 13.92 μM) either singly or in the following combinations (IAA + BA; IAA + Kin, and BA + Kin) produced granular callus except BA + Kin which resulted in compact, hard, greenish-white (CHGW) callus. The optimum CHGW callus (2.62 g fresh weight/ explant) was produced on MS media with 13.32 μM BA + 2.32 μM Kin with over 93% callus induction frequency. Optimum shoot organogenesis (67% frequency) was achieved in CHGW callus with lower level of BA (2.22 μM) and Kin (2.32 μM) and produced 3.2 shoots/0.5 g callus within 35 d of culture. Microshoots were rooted successfully (62% frequency) after 35 d of culture on 1/2MS containing 4.90 μM IBA and hardened off. Antioxidant enzymes such as catalase, peroxidase, polyphenol oxidase, and biochemical parameters viz. hydrogen peroxide, reducing and nonreducing sugars, starch, proteins, phenols, and proline contents were studied in regenerating and nonregenerating CHGW calluses to establish a correlation between these parameters and shoot morphogenesis. All the enzyme activities and biochemical parameters were found more in regenerating callus than in nonregenerating except phenols.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghums

Callus and plant regenertion were induced from shoot portions of mature embryos (dry seeds) of five high tannin Sorghum bicolor (L.) Moench cultivars. The explants were cultured on Murashige and Skoog medium with altered concentrations of 5 salts, supplemented with 150 mg/L L-asparagine, 5mg/L 2,4-Dichlorophenoxyacetic acid and 0.05mg/L kinetin. Calli which were yellow and globular were formed with 70–90% frequencies. The subculture medium which gave best results was MS with 2mg/L 2,4-Dichlorophenoxyacetic acid and 0.5mg/L kinetin. Plants were regenerated on MS medium supplemented with 150mg/L L-asparagine and 0.2mg/L kinetin with regeneration frequencies of 11–48%.Abbreviations 2,4-D dichlorophenoxyacetic acid     

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.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

Sodium nitroprusside enhances callus induction and shoot regeneration in high value medicinal plant Canscora diffusa

Plant Cell, Tissue and Organ Culture (PCTOC) - In different countries, mostly in EU, rules for strawberry nursery propagation impose the use of micropropagation only to produce stock virus free...     

Somatic embryogenesis and plant regeneration of turf-type bermudagrass: Effect of 6-benzyladenine in callus induction medium

In order to optimize tissue culture conditions for bermudagrass, an important warm-season turfgrass species, tissue culture responses of young inflorescences of a hybrid bermudagrass cultivar `Tifgreen' (Cynodon dactylon×Cynodon transvaalensis) and a common bermudagrass cultivar `Savannah' (Cynodon dactylon) were investigated. When cultured on Murashige and Skoog medium with 4.52 to 13.57 μM (1–3 mg l^-1) 2,4-D, young inflorescence segments yielded non-embryogenic calli which were unorganized and had loosely associated, long tubular cells on the surface. However, inclusion of 6-benzyladenine (BA) in callus induction medium at a level of 0.044 μM (0.01 mg l^-1) induced formation of a compact, nodular embryogenic structure on approximately 20% of the calli. Calli with such a compact embryogenic structure were highly regenerable. When young inflorescences smaller than 0.75 cm were cultured, the embryogenic structure yielded green plantlets with regeneration rates of 79.5% and 83.3%, respectively for the two cultivars. All 96 plants regenerated from calli induced in the BA-containing medium were green and morphologically normal. The embryogenic nature of the compact structure was confirmed by scanning electron microscopy. This revised version was published online in June 2006 with corrections to the Cover Date.