Naphthoquinone contents of in vitro cultured plants and cell suspensions of Dionaea muscipula and Drosera species

In vitro cultured carnivorous plants were grown on a hormone-free medium. They produced the following naphthoquinones: Dionaea muscipula (plumbagin: 5.3%), Drosera rotundifolia (7-methyljuglone: 0.6%), D. binata (plumbagin: 1.4%), and D. capensis (7-methyljuglone: 0.5%). A red, slow-growing suspension culture of D. muscipula was maintained in a modified McCowns Woody Plant (McC) medium and produced plumbagin (2.59%) after 30 days growth. A suspension culture of D. rotundifolia grew slowly as multicoloured small aggregates only in a modified Murashige and Skoog (MS) medium. No quantifiable amounts of naphthoquinones were produced. Several cell lines of D. capensis were developed. Green aggregates grown in a modified MS medium contained 7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink cultures grown in modified McC medium contained 7-methyljuglone (1.24%), while dark red cultures produced ca. 1% in both modified McC and MS media. Though the latter medium was significantly better with regard to biomass production, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 g dry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showed that there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.     

Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solanum tuberosum L

Freshly isolated mesophyll and suspensions-cell protoplasts of S. tuberosum cvs. Desiree and Maris Piper were cultured in different media i.e. modified MS, V-KM and MS-KM. Protoplast plating efficiencies were higher in MS-KM medium. Resulting protoplast-derived calluses were transferred either onto the medium of Bokelmann and Roest (1983) or that of Lam (1977) for shoot regeneration. Calluses derived from mesophyll cell protoplasts differentiated about 2 weeks earlier than calluses derived from suspension-cell protoplasts. Shoot initiation was also about 2 weeks earlier from calluses subcultured onto the former medium as compared to the latter.     

Protoplast culture and somaclonal variability of species of series Juglandifolia

Mesophyll protoplasts of species of series Juglandifolia (Solanum rickii, S. lycopersicoides, S. ochranthum and S. juglandifolium) were isolated and cultured in liquid nutrient media TM-2 or KM8P. The cell colonies formed were transferred onto agar-solidified media TM-3 or GM, and 10 to 15 days later onto TM-4, PRM, MS3ZG, KK or C regeneration media. Formation of the shoots for S. rickii and S. lycopersicoides was observed after 30 to 35 days on regeneration medium. The regenerated shoots were rooted on hormone-free MS medium. Morphological and cytogenetic analyses have shown that somaclonal variants might arise in the course of plant regeneration from protoplasts of these species.Abbreviations BAP 6-benzylaminopurine - 2, 4-D 2, 4 dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - B5 Gamborg medium - TRIS tris(hydroxymethyl)aminomethane     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.     

A protoplast to plant system for the chrysanthemum Dendranthema zawadskii x D. grandiflora

Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - MS Murashige and Skoog (1962) - NAA 1-naphthylacetic acid - Pfr Photon fluence rate - V-KM Binding and Nehls (1977)     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.     

Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var ‘capitata’)

Summary Protoplasts were enzymatically isolated from the first leaves of cabbage (Brassica oleracea var capitata, F1 hybrid Baochun). Sustained cell division and somatic embryogenesis were obtained after culturing the protoplasts in modified liquid DPD medium supplemented with CaCl₂ · 2H₂O 800 mg/l, 2,4-D 0.5 mg/l, kinetin 1 mg/l, 0.3 M mannitol and sucrose 20 g/l. Upon transferring cell colonies onto a modified Murashige and Skoog (MS) agar medium, small calli were gradually formed. Callus proliferated on MS medium supplemented with hormone combinations of 2,4-D 0.1–0.5 mg/l and kinetin 3–4 mg/l. Multiple shoots were induced on differentiation medium supplemented with 3 mg/l of kinetin and 0.1 mg/l of gibberellic acid GA₃. After transferring differentiated shoots onto MS medium supplemented with indoleacetic acid (IAA), kinetin, GA₃ at 0.1 mg/l each and 500 mg/l of N.Z. amine, intact plants were eventually produced.     

Plant regeneration via somatic embryogenesis from suspension cell cultures of Lilium×formolongi hort. Using a bioreactor system

Summary In vitro seedlings of Lilium × formolongi Hort. evs. Norikula, RaiZen No. 1, RaiZen No. 3, RaiZen Early, and Bailansa were used to induce callus by variously modified Murashige and Skoog (MS) media, using protocols for flask culture and bioreactor culture. Green embryogenic callus proliferated from roots near the base of bulblets of five varieties on media containing 0.53–5.3 μM α-naphthaleneacetic acid (NAA), and 28 cell lines were obtained by subcultures on the same medium. For flask culture, the fresh weight (FW) of embryogenic cell clumps doubled every 4 wk on MS basal salts supplemented with 0.53°M NAA and 30 g l⁻¹ sucrose. The maximum frequency of somatic embryos that developed into plantlets was 76.67±17% when plated onto solid MS basal medium without plant growth regulators (PGRs). Among the treatments using four types of bioreactors, the best cell growth and regeneration rate (74±0.14%) of somatic embryos was in a modified 2–1 bioreactor. Cells incubated in the other three bioreactors furned brown and died. Histological study revealed that regeneration was by somatic embryogenesis. The regenerants showed normal growth and flowering after 8–9 mo, in the field. A cell line of cv. Norikula has been subcultured in MS basal salts containing 0.53 μM NAA every 2 mo. for 6 yr. The cell aggregates became more synchronous and many typical embryogenic cells with dense cytoplasm were observed under a light microscope. The long-term embryogenic cells plated on MS basal medium still gave rise to numerous somatic embryos and converted into plantlets.     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

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 plant regeneration of Ilex paraguariensis (aquifoliaceae)

Summary Nodal segments as well as shoot tips and apical meristems of 2-yr-old “maté” plants (Ilex paraguariensis St. Hil.) were cultured in vitro to establish micropropagation systems. Maximum shoot regeneration was achieved when nodal segments were cultured with 1/4 Murashige and Skoog (MS) medium with 3% sucrose. We induced roots to differentiate by transferring the regenerated shoots onto the same medium, solidified with 2.5 g Phytagel per 1 and supplemented with indole-3-butyric acid (7.4 μM) and finally transferring shoots to 1/4 MS medium with 3% sucrose and lacking growth regulators. Plants were successfully established in soil.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd.

In vitro propagation was initiated from 2-week-old and 7-month-old explants of Acacia mangium. Juvenile explants (2 week-old) of 5- to 10-mm lengths composed of two leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 or 2.0 mg L^-1 6-benzyladenine (BAP). After 6 weeks, most explants had formed a large cluster of 14–18 axillary shoots produced by prolific branching of the primary axillary shoot after elongation. The maximum multiplication rate (40) was obtained in the first subculture; the rate decreased to 10–20 in the second one. The mean length of shoots was not significantly affected by BAP concentrations during the subsequent cultures. Rooting ability of juvenile explants was greatly affected by BAP concentrations used in the multiplication medium. When both types of explants were multiplied on a MS medium containing 1.0 mg L^-1 BAP and transferred to a half-strength MS medium containing 0.05 mg L^-1 IBA, only 10% of the juvenile explants were rooted versus 70% of the 7-month-old explants. Rooted plants transferred onto artificial substrate were all nodulated, when inoculated with a specific Bradyrhizobium sp. strain.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Selection of Daucus cybrids based on metabolic complementation between X-irradiated D. capillifolius and iodoacetamide-treated D. carota by somatic cell fusion

Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion.     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.