Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha citrata were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using ³²P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.Abbreviations aux 1 Tryptophan monoxygenase - aux 2 Indoleacetamide hydrolase - ipt Isopentenyl transferase - tzs Trans-Zeatin secretion locus - 2,4-D 2,4-Dichloro phenoxyacetic acid - oct Octopine - nop Nopaline - suc L,L-succinamopine - T-DNA transferred DNA     

Propagation of Costus speciosus (Koen.) Sm. through in vitro rhizome production

Rhizomes developed in vitro on shoots of Costus speciosus (Koen.) Sm. which were initiated from zygotic embryos. The effect of different hormonal and nutritional additions to Schenk and Hildebrandt' s (SH) basal nutrient medium on rhizome development was studied. Rhizomes developed on SH basal salts but formed with increased frequency on medium supplemented with adenine sulfate, casamino acids (CA) and various combinations of cytokinins and auxins. Incubation in light was necessary for rhizome formation. Isolated basal as well as nodal (aerial) rhizomes formed and produced new shoots. In basal medium without any growth additives (control) the average number of shoots produced per inoculum was 3.3 ±0.73 whereas in the best suited medium i.e. supplemented with 250 mg l^–1 CA the number of shoots obtained was 22.7±1.92. There was no dormancy period for rhizomes under the cultural conditions investigated. Rhizome-sprouts were easily transplanted from the in vitro conditions to the field. More than five hundred propagules (i.e. sprouted-rhizomes) were obtained within 5 months and it has been estimated that more than 2.4 × 10⁵ propagules could be achieved per year through four subsequent in vitro rhizomes-generation cycles. Thus, a rapid method of propagation has been established.Abbreviations AdS Adenine sulphate - BA benzyladenine - CA casamino acids (vitamin free) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn Kinetin - NAA naphthaleneacetic acid - SH Schenk and Hildebrandt's medium (1972)     

Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson

Prolific shoot bud differentiation was induced in callus and suspension cultures of hypocotyl origin in Brassica campestris cv. Yellow Sarson on MS medium supplemented with K (13.9–23.2 M) or BA (13.3–22.1 M). Plantlets were obtained by rooting the in vitro differentiated shoots. Histological studies revealed a unique mode of meristemoid formation.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA Benzyladenine - IAA Indole-3-acetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthalene acetic acid     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Somaclonal variation in the berberine-producing capability of a culture strain of Thalictrum minus

Variation in the productivity of berberine between clonal cell lines derived from a culture strain of Thalictrum minus. var. hypoleucum was investigated during the period of successive transfer generations. There was a correlation between berberine productivity and growth in these clonal cultures. Although no significant difference was found in the secretory function as well as the qualitative pattern of alkaloids, there was wide variation in the yield of berberine among the clones. Some of the cell lines have shown a high stability throughout the successive subculturing, whereas the other lines tended to either decrease or increase the productivity continually before they have become more or less stable at low or high levels. After eight months of subculturing, one of the high-producing cell lines yielded 0.76 g of berberine per liter in 14 days of culture strain. In view of variations observed in both primary and secondary clones, the possible cause of the quantitative variation in berberine production in Thalictrum cultures is discussed.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine     

A parenchymatic medium solidifier for plant in vitro culture

A new material for the solidification of liquid culture media was prepared from plant parenchyma tissues by mechanical subdivision, solute extration and dessication from ethanol. It is suitable for in vitro culture and propagation of callus as well as shoot tip cultures. The following plant materials have been grown by means of the new medium solidifier: shoot cultures of Betula pendula Roth, Gerbera jamesonii H. Bolus ex Hook and Floribunda rose "Triumph", callus tissues of Daucus carota L. and Chenopodium album L. The new solidifying material has special advantages over agar for application in the rooting phase of in vitro propagation.Abbrevations PMS parenchymatic medium solidifier - MS Murashige and Scoog's medium - IAA Indole-3-acetic acid - B biotin - K kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - ch caseine hydrolysate     

In vitro propagation and low temperature storage ofSaussurea lappa C.B. Clarke — An endangered,medicinal plant

A procedure forin vitro multiplication ofSaussurea lappa (Asteraceae) is described. On Murashige and Skoog's medium (MS) containing benzylaminopurine and gibberellin 3.5-fold shoot multiplication occurred every three weeks. Shoots rooted on MS containing 0.5 M naphthaleneacetic acid with 90% efficiency. The shoot cultures stored at 5°C in the dark for 12 months without an intervening subculture survived with 100% viability. The shoots cold stored for 6 months or more showed higher rates of multiplication under culture room conditions than the untreated shoots.Abbreviations MS Murashige and Skoog 1962 - BAP Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - GA₃ Gibberellin     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt     

Transformation of Medicago by Agrobacterium mediated gene transfer

Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.Abbreviations Km kanamycin - KmR kanamycin resistant - Cb carbenicillin - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine - T-DNA transferred DNA into plants     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.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 - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

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     

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)     

Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014%), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species.Abbreviations B₅ medium after Gamborg et al. (1968) - 2,4-D 2,4, dichlorophenoxyaceticacid - MS medium after Murashige and Skoog (1962) - BAP benzylaminopurine - IBA indole-3-butyric acid - Kn kinetin - CH casein hydrolysate - T-DNA transferred DNA     

Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens

Summary To determine the best combination for potential use in transformation of Pisum sativum L., 13 genotypes were inoculated with wild-type Agrobacterium tumefaciens strains A281, C58 and Ach5. A281 appeared to be the most virulent strain, as determined by size and number of tumours, followed by C58 and Ach5. Genotypes differed considerably in their response to inoculation and genotype x strain interaction was evident. Genotypes also responded differently to in vivo or in vitro inoculation. Axenic calli from tumours could be grown on hormone-free medium and the presence of the specific opines for each strain in the callus indicated successful transfer and expression of T-DNA. Southern blot analysis of DNA from callus of A281-inoculated material showed that both T_R and T_L T-DNA had been incorporated into the pea genome.NRCC No. 30265     

The presence of concanavalin A and canatoxin in Canavalia ensiformis DC tissue culture

Isolated embryos, cotyledons and embryos plusa fragment of cotyledon from seeds of Canavalia ensiformis (jack bean) were cultured in vitro. Concanavalin A and canatoxin cross-reactive material were detected by double immunodiffusion tests. Canatoxin was detectable until 30 days in cultures of embryos, embryos plus cotyledons and hypocotyls. Concanavalin A was also present in all cultures being detected until 90 days in cultures treated with 6-benzylaminopurine. No concanavalin A was detected in root cultures. Concanavalin A was present in cell suspensions until 45 days of culture; the culture medium contained neither concanavalin A nor canatoxin. Tissue cultures thus can produce Con A and CNTX and will be an important research tool for studying the biosynthesis of such substances.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - CNTX canatoxin - Con A concanavalin A - CRM cross-reactive material - DEAE-cellulose diethylaminoethyl-cellulose - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris tris (hydroxymethyl) aminomethane     

Plant regeneration from protoplasts of common buckwheat (fagopyrum esculentum)

Protoplasts were isolated from hypocotyls of etiolated seedlings from a diploid and the corresponding autotetraploid variety of common buckwheat (Fagopyrum esculentum). The isolated protoplasts started to divide after 4 days in culture in a modified MS medium. Maximum plating efficiency was approximately 1%. Regenerated calli derived from the tetraploid genotype developed roots easily but were recalcitrant to form shoots. Eighteen months following the initiation of cultures, tetraploid embryoids and shoots emerged after 3 weeks on an MS medium containing 0.1 mg/l gibberellic acid.Abbreviations 2,4-D 2,4 — dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - GA gibberellic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone