Micropropagation of Alpinia purpurata from inflorescence buds

Alpinia purpurata K Schum inflorescence buds inoculated on Murashige and Skoog medium (MS) containing 10 M 6-benzyladenine with 5 M naphthaleneacetic acid gave rise to multiple shoot formation with a mean increase of 15 to 20 new shoots each 4 weeks. Plants could be stored for more than 6 months in flasks containing deionized water with 5 g 1^-1 sucrose with minimal vegetative growth.Abbreviations IAA indole-3-acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - AS Adenine sulphate - MS Murashige and Skoog medium     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA ascorbic acid - AC activated charcoal - CA citric acid - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - KC Knudson-C (1946) basal medium - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & McCown 1980)     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture

A procedure for rapid in vitro propagation of the aromatic and medicinal plant Hemidesmus indicus (L.) R.Br. (Family Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate of 8.2 ± 0.4 shoots/explant with a 95% frequency was achieved in S weeks culture period on Murashige and Skoog medium supplemented with 1.15 M kinetin and 0.054 M -naphthaleneacetic acid. Excised shoots were rooted on the same basal medium supplemented with 1.15 M kinetin and 7.35 M indole-3-butyric acid. Shoots derived from subcultures exhibited better rooting response than those from primary cultures. After a hardening phase of 2 weeks, there was a 70% transplantation success in the field.Abbreviations MS Murashige and Skoog (1962) medium - BA N⁶ benzyladenine; KN kinetin - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 g/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 g/ml kanamycin, but exhibited reduced rooting ability.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NPT-II neomycin phosphotransferase(EC 2.7.1.95) - GUS -glucuronidase(EC 3.2.1.31) - X-GLUC 5-bromo-4-chloro-3-indolyl-glucuronide - 4-MU 4-methylumbelliferone NRCC No. 31227     

Regeneration of plants from callus tissue of Aeschynomene spp. (Leguminosae)

Plants were regenerated from leaflet-derived callus of Aeschynomene sensitiva, A. americana and A. villosa. Explants were induced to form callus when aseptically cultured on Murashige and Skoog medium solidified with 0.8% agar and containing 0.5 or 0.05 M naphthaleneacetic acid and 4.4 or 13.3 M benzyladenine. Shoot regeneration was readily achieved. Roots were induced when shoots were transferred to medium devoid of growth regulators or with 0.05, 0.5 or 5.4 M naphthaleneacetic acid. Plantlets were successfully transplanted to soil. Callus from A. falcata failed to regenerate shoots. Explants from leaflets of A. fluminensis did not produce callus when cultured in vitro.Abbreviations BA benzyladenine - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid     

In vitro regeneration of plantlets from shoot callus of mature trees ofDalbergia latifolia

A successful procedure was established for in vitro mass multiplication of Indian rosewood (Dalbergia latifolia Roxb.). In vitro regeneration of plantlets was achieved from callus of shoot tips and shoot segments of over 50-year-old elite trees on Murashige & Skoog's medium containing naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). For rooting, regenerated shoots from the calli were excised and first treated with White's liquid medium or half-strength Murashige & Skoog's medium, supplemented with indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid for 48 h to 72 h. Following this treatment, plantlets were transferred to hormone-free half-strength MS medium. Rooted plantlets were then transferred to pots and grown in the greenhouse.Abbreviations BAP 6-benzylamino pruine - CH casein hydrolysate - CM coconut milk - 2, 4-D dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PVP-10 polyvinyl pyrrolidone - YE yeast extract     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

Preliminary investigations on somatic embryogenesis from leaf discs of red oak (Quercus rubra L.)

Somatic embryos were obtained from leaf discs of juvenile red oak plants. Basal inductive nutrient medium was a modified Murashige and Skoog solution enriched with 500 mg L^–1 casein hydrolysate, 100 mg L^–1 polyvinylpyrrolidone, 5.4 M naphthaleneacetic acid and 0.09 M benzyladenine. Embryogenesis was obtained only from leaf discs in the presence of light and increased when the adaxial surface of the explants (with midrib or main veins present) was in contact with the medium. Large variation was observed in all experiments. Recurrent embryogenesis was observed at the base of embryo clusters with callus present; conversely, embryogenic potential was rapidly lost by subculturing full calli. Maturation, germination and development of isolated somatic embryos were obtained. However, the vast majority of embryos did not have viable apical bud meristems and on only a few occasions were shoots produced.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2iP isopentenyladenine - NAA naphthaleneacetic acid - 2.4-D 2.4-dichlorophenoxyacetic acid - GA gibberellic acid - PVP polyvinylpyrrolidone     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

In vitro culture ofXanthium strumarium (Cocklebur)

Xanthium strumarium L. was micropropagated by rooting shoots proliferated from shoot-tip explants. The best shoot proliferation was obtained from explants growing on Murashige and Skoog medium supplemented with 4.4 to 8.9 M benzyladenine (BA) and 1.1 to 2.1 M naphthaleneacetic acid (NAA). The micropropagated plants were transferred to potting media and maintained under high humidity conditions in the greenhouse. The media that produced best shoot proliferation from shoot-tip explants also produced the most callus from hypotocotyl, cotyledon and shoot-tip explants, whereas more callus was produced on leaf explants with a lower BA concentration (1.1 M) and 1.1 M NAA.Abbreviations BA benzyladenine, 2 4-d-2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog Technical contribution No. 3319 of the South Carolina Agricultural Experiment Station, Clemson University.     

Micropropagation of Phytolacca dodecandra through shoot-tip and nodal cultures

A procedure for micropropagation of endod (Phytolacca dodecandra) is described. BA at 0.44 M produced 3.1 new shoots per expiant in six weeks using shoot tips. Nodal expiants, however, produced up to 4.7 shoots per explant on medium with 0.44 M BA and 0.27 M GA,. IBA at 0.49 M induced 90% rooting with minimal callus. Plantlets were successfully transferred to the greenhouse and some staminate clones produced flowers after six months.Abbreviations BA 6 benzylaminopurine - kinetin 6-furfurylaminopurine - 2iP N⁶-(2-isopentyl)adenine - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - GA₃ Gibberellic acid - IBA indole-3-butyric acid