In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

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)     

Release and crystallization of berberine in the liquid medium of Thalictrum minus cell suspension cultures

Cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. were found to produce a large amount of berberine (400–800 mg/l) when 5–10 M 6-benzyladenine was added to Linsmaier and Skoog's medium containing 100 M 1-naphthaleneacetic acid. Most of the berberine produced was released continuously from the cells into the liquid medium, and an excess of berberine crystallized as its nitrate in the medium. When the cells were cultured in a modified LS medium containing 20 mM KNO₃ and 40 mM NH₄Cl in place of 20.6 mM NH₄NO₃ as nitrogen source, most of the alkaloid crystallized to form berberine chloride instead of nitrate. Minor alkaloids, thalifendine and magnoflorine, were also isolated from the medium and identified.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine     

Anthocyanin production in callus cultures of roselle (Hibiscus sabdariffa L.)

Callus tissues derived from seedlings of roselle (Hibiscus sabdariffa L.) were shown to produce two cyanidin glycosides as major anthocyanin pigments. Both callus growth and anthocyanin synthesis were remarkably stimulated by 2,4-dichlorophenoxyacetic acid. The highest anthocyanin yield was observed when 1 M 2,4-D in combination with 0.1–1 M kinetin was supplemented to the culture medium. In contrast, gibberellic acid showed inhibitory effect on anthocyanin production.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - GA gibberellic acid     

Somatic embryos from callus of Sequoia sempervirens

Compact calli with a potential for somatic embryogenesis were obtained from complete or split mature zygotic embryos or from cotyledons and hypocotyls of in vitro grown seedlings of Sequoia sempervirens. Somatic embryos which showed a typical bipolar structure, were formed together with adventitious buds. When placed on filter paper supports they developed into complete plantlets. Of the various combinations tested, culture medium adapted from Murashige and Skoog mineral solution complemented with 6-benzylaminopurine (2 M), kinetin (2 M) and 2,4-dichlorophenoxyacetic acid (2.5 M) was established as the optimal for somatic embryo production.Abbreviations BM basic medium - 2,4-D dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kin Kinetin     

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)     

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

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

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Influence of plant growth regulators,basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law.) cotyledon explants

Applications of in vitro screening techniques for Pinus ponderosa resistance to Peridermium harknessii could be beneficial in a tree breeding program. Plant growth regulators, basal media formula and carbohydrate levels were examined to determine the various effects each would have on excised cotyledon growth and development. Proliferating green callus was initiated from cotyledon explants on SH basal medium containing 4.4 M BA:5.4 M NAA and 1% sucrose. Subsequent subculturing onto LS medium supplemented with 44.0 M BA: 5.4 M NAA and 2% sucrose improved callus maintenance. The highest frequency of caulogenesis from cotyledon explants occurred on a modified GD medium containing 44.0 M BA: 0.054 M NAA and 4% glucose. The influence of nitrogen source, osmoticum and medium salt concentrations are discussed relative to callus initiation and caulogenesis.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - CD Campbell & Durzan - GD Gresshoff & Doy - LP Le Poivre - LS Linsmaier & Skoog - MC McCown - SH Schenk & Hildebrandt     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium     

Shoot regeneration from petioles and leaves of Vitis X labruscana ‘Catawba’

Summary Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana Catawba. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.0–10.0 M BA and 0.1–0.5 M IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NN69 Nitsch and Nitsch (1969) medium - NOA 2-naphthoxyacetic acid     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plantlet regeneration from petiole explants of the african horned cucumber,Cucumis metuliferus

Plantlet regeneration in Cucumis metuliferus from several explant sources, including cotyledons, leaves, hypocotyls and petioles, was evaluated on Murashige and Skoog's medium containing various combinations of auxin (IAA, NAA, 2,4-d) and cytokinin (BA, kinetin, zeatin), Callus development was obtained within 4 to 5 weeks on all growth regulator combinations which were tested at concentrations ranging from 1.0 M to 4.0 M of each. The response was similar when the tissues were incubated under light or in continuous darkness. Differentiation of callus to form adventitious buds or shoot primordia occurred only with petiole explants on medium containing NAA/BA or 2,4-d/BA at 2.0/1.0 M; none of these calluses, however, differentiated further to form shoots. When the differentiated calluses derived from petiole explants which had been initiated on 2,4-d/BA at 2.0/1.0 M were transferred onto medium with 2.0 M zeatin, formation of shoots occurred within 2 to 3 weeks. The frequency of shoot formation was 14.6%. Subculture of these shoots onto MS medium without growth regulators gave rise to plantlets of normal appearance. Regeneration in C. metuliferus requires callus initiation on an appropriate growth regulator regime followed by transfer to a medium containing the cytokinin, zeatin, and can be achieved within 10–12 weeks.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plantlet regeneration from mature zygotic embryos of eastern white pine (Pinus strobus L.)

Plants were regenerated from whole embryo explants obtained from eastern white pine (Pinus strobus L.) seeds. Embryos were surgically removed and axenically cultured to induce buds in vitro on a modified Murashige and Skoog medium containing various concentrations of 6-benzylaminopurine. Embryos remained on bud induction medium for 21 days and then were transferred to the same basal medium without 6-benzylaminopurine to promote bud development and subsequent shoot elongation. The medium containing 10 M 6-benzylaminopurine induced the greatest number of shoots per embryo. Rooting was achieved by direct transfer of the shoots to a non-sterile artificial soil mixture followed by multiple treatments with 15 nM 1-naphthaleneacetic acid. Regenerated seedlings are currently growing under greenhouse conditions.Abbreviations NAA 1-napthaleneacetic acid - BA 6-benzylaminopurine - SIM shoot induction medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA Triiodobenzoic acid - 2iP 2-isopentenyl adenine     

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 M NAA and 10 M BAP, and embryo maturation was promoted after transfer to a medium containing 1 M NAA and 10 M BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 M NAA and 1 M BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 M NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.Abbreviations BAP 6-benzylaminopurine - B5 medium of Gamborg et al. 1968 - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium of Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid     

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     

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.