Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.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 - BAP 6-benzylaminopurine     

Somatic embryogenesis in quite a direct way in cultures of mesophyll protoplasts of Brassica napus L.

Somatic embryos and plantlets have been obtained in quite a direct way from mesophyll protoplasts isolated from androgenetic plants of two cultivars (Loras and Tower) of Brassica napus. The procedure consists of four steps. Proembryos were induced in a medium supplemented with a cytokinin and an auxin at comparatively high concentrations. They developed to mature embryos when the auxin was reduced or omitted.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA 1-naphthaleneacetic acid Dedicated to Prof. Dr. Wilhelm Halbsguth on the occasion of his 70th birthday.On leave from the Institute of Genetic, Academia Sinica, Beijing, China     

Regeneration in Phaseolus vulgaris L. Promotive role of N6-benzylaminopurine in cultures from juvenile leaves

Plants were regenerated from cultured young leaves of Phaseolus vulgaris L. cv. Kinghorn. For inducing shoot regeneration the expiant had to consist of the petiole and a portion of the lamina, and N⁶-benzylaminopurine (BAP) had to be present in the culture medium. Furthermore, the frequency of shoot regeneration increased more than seven-fold if donor seedlings were raised on a medium containing 5 M BAP, followed by culture of the leaf explants on a medium containing 20 M BAP. Regenerated shoots developed roots on basal (hormone-free) medium and the resulting plantlets could be transplanted to soil.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium This research was supported by operating grants from the Research Board Grants Program of the University of Guelph and the Natural Sciences and Engineering Research Council of Canada to PKS. Technical and photographic assistance from Sangeeta Saxena and Jean Gerrath is gratefully acknowledged.     

Selective enhancement of Ipomoea batatas Poir. embryogenic and non-embryogenic callus growth and production of embryos in liquid culture

Embryogenic callus cultures of Ipomoea batatas Poir. produce fast growing non-embryogenic material which soon dominates the cultures. Our objective was to selectively enhance the proliferation of the embryogenic fraction. For this, the effect of BAP and 2,4-D concentrations on growth of embryogenic and non-embryogenic callus were studied and consequently, nutrient media for the production and indefinite maintenance of embryogenic callus without embryo formation were defined. Selective proliferation of embryogenic callus was obtained on solid media with 10 M 2,4-D and 1 M BAP and in liquid media with 5 M 2,4-D. Selective proliferation of non-embryogenic callus occurred in liquid medium with 1 M 2,4-D. In embryogenic liquid culture, embryos were produced with 0–2 M 2,4-D. Increasing 2,4-D concentration from 0 to 2 M in these cultures restricted embryo development.Abbreviations 2,4-D = 2,4-dichlorophenoxyacetic acid - BAP = 6-benzylaminopurine     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

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)     

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     

Propagation of Casuarina equisetifolia through axillary buds of immature female inflorescences cultured in vitro

The study of the actinorhizal symbiosis in Casuarina equisetifolia requires an homogenous plant material. Consequently, we devised a method of micropropagation based on the use of immature female inflorescences (IFI) as explants. IFI excised from an adult tree formed multiple buds after 4-week incubation on Murashige and Skoog medium with 0.05 mol 1^–1 NAA and 11.1 mol 1^–1 BAP. The axillary buds evolved into 5–6 cm long shoots 5 weeks after the transfer of IFI on a similar medium except for the addition of activated charcoal. Rooting of the shoots was obtained on a third medium, without BAP or charcoal, but with 1 mol 1^–1 NAA. The plantlets were transferred into soil. Their growth was satisfactory and no plagiotropic tendency was observed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - IFI immature female inflorescences - NAA -naphtaleneacetic acid     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. ‘Moneyway’

Plants were regenerated from excised adventitious roots of the rose rootstock Moneyway via a three step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced on excised roots after incubation for 4 weeks in the dark on SH-medium (Schenk and Hildebrandt) containing 50 M 2,4-dichlorophenoxyacetic acid. For embryo induction, calluses were transferred to hormone-free SH-medium and incubated for 8 weeks. The use of Gelrite instead of agar during callus induction stimulated somatic embryogenesis (up to 16% of the explants formed organized structures), whereas the presence of 6-benzylaminopurine in this phase inhibited subsequent regeneration. Yellow solid calluses with embryo-like cotyledons or primordia and friable calluses with embryos were selected, and upon incubation in the light shoots developed. Shoot development was faster and more frequent on solid callus than on friable callus (64% and 21% of the calluses finally formed one or more shoots, respectively). Eleven out of thirteen regenerants developed similarly to control shoots. Finally this regeneration method is compared with other systems for somatic embryogenesis and opportunities for the production of transgenic rose rootstocks and rose cultivare are discussed.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BM+ enriched basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI-4,6 diamidino-2-phenylindole - FeEDDHA ferric ethylenediamine di(ohydroxyphenylacetate) - FeEDTA ferric ethylenediamine tetraacetate - IBA indole-3-butyric acid     

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     

In vitro plant regeneration from leaf-derived callus in ginger (Zingiber officinale Rosc.)

Excised tissues from young leaves of ginger cv. Maran were cultured on revised Murashige and Skoog medium supplemented with various concentrations of growth regulators. The presence of 2, 4-D in the culture medium at 9.0–22.6 M resulted in callus growth. Organogenesis and plantlet formation occurred when the concentration of 2,4-D is reduced to 0.9 M and with the addition of 44.4 M BA into the medium. The rate of plant regeneration increased when the growth regulators are completely removed from the culture medium in the subsequent subcultures. The plantlets developed extensive root systems when they were put in MS liquid medium with 5.4 M of NAA. The establishment of these plantlets in soil is about 80%.Abbreviations BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar Centennial on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. Centennial expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - GUS -glucuronidase - IAA indole-3-acetic acid - NPT II neomycin phosphotransferase II     

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     

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     

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     

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoog's semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 M indoleacetic acid and 0.046 M kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.Abbreviations MS Murashige and Skoog's medium - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - RH relative humidity     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Somatic embryogenesis from mesophyll protoplasts of Trigonella corniculata (leguminosae)

Mesophyll protoplasts isolated enzymatically from Trigonella corniculata divided to form callus, with a plating efficiency of 49% in Kao (1977) medium. Protoplast-derived tissues formed somatic embryoids at high frequency on MS medium with 2.0 mg L^–7 NAA and 0.5 mg L^–7 BAP. Embryoids developed into plants on MS medium lacking hormones.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - MES 2-(N-morpholine) ethanesulphonic acid - NAA -naphthaleneacetic acid On leave from Genetics Institute, Academia Sinica, Beijing, China