An Investigation of Factors which Influence the Production of Abnormal Terpenoids by Callus Cultures of Andrographis paniculata Nees

Callus and suspension cultures of Andrographis paniculata havebeen shown to produce three new sesquiterpene lactones whichhave been named paniculides. A, B, and C and not the andrographolides(diterpenes) produced by the intact plant. Cultures derived from leaves, stems, hypocotyls, roots, andembryos accumulated paniculides, but no andrographolides weredetected. Conversely andrographolides, but no paniculides weredetected in extracts from leaves, stems and roots of intactseedlings. Modifications of the medium including the use of different syntheticauxins and the replacement of coconut milk with kinetin andcasein hydrolysate did not influence paniculide production bythe hypocotyl cultures. In addition an established root isolatecultured for 9 months on a simple synthetic medium without auxin,meso-inositol or coconut milk still synthesized significantquantities of paniculides. Diurnal fluctuations of temperature and light did not influencethe pattern of compounds produced although cultures grown incomplete darkness produced the individual paniculides in differentproportions. Possible explanations for these findings are discussed.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Effects of Auxin and Kinetin on the Development and Differentiation of Potato Tissue Cultured in vitro

The present investigation deals with the influence of auxin and kinetin on cultures of potato tuber tissue, and the effect of the size of the explants on the organ formation. Exogenous application of auxin is indispensable for initiating callus formation of the tissue, but kinetin is not necessarily required. Kinetin serves to maintain the callus development, indicating that the stimulation of callus growth due to exogenous auxin would presumably be mediated by the addition of kinetin to the medium. A relatively low concentration of auxin in the medium is required for root formation on the culture. In this case, IAA is markedly superior to any other auxin in root forming activity. NAA is somewhat less effective than IAA, and 2,4-D shows no stimulation on root formation. The growth and organ formation of potato tissue are remarkably affected By the variation in sizes of the explants. Spontaneous formation of a sprout was observed on the large explant in the medium without auxin. Small explants form only callus or roots in the presence of auxin and kinetin irrespective of combination or concentration. It seems likely that a sufficient amount of stimulus for initiation of bud formation may be contained in the large sized explant itself.     

Morphogenesis in callus tissue cultures of someMatricaria andAchillea Species

In the present paper we deal with the possibility of morphogenesis induction in callus tissue cultures of some representatives ofMatricaria andAchillea species. Shoot regeneration from calli ofMatricaria chamomilla andM. inodora has been induced by 0.1 mg l^-1 kinetin or by combination of 0.5 mg l^-1 kinetin and 0.5 mg l^-1 NAA added to Murashige-Skoog culture medium. Rhizogenesis took place without any other addition of auxin. In callus tissue cultures ofAchillea ptarmica cultivated on Murashige-Skoog medium with 1 mg l^-1 2,4-D after a year long cultivation the whole plant has been regenerated without any change of nutrient requirements. In callus tissue ofA. nobilis under the same conditions only roots wore regenerated.     

Co-inoculation of Ceratocystis polonica and Heterobasidion annosum with callus of two Norway spruce clones with different in vivo susceptibility

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorophenoxyacetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation ofCereus peruvianus mill. (cactaceae)

Summary Cereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

Induction of Callus from Flesh of Gardenia jasminoides ELLIS Fruit and Formation of Yellow Pigment in the Callus

Callus cultures were derived from flesh sections of young fruit of Gardenia jasminoides ELLIS on Murashige-Skoog agar medium supplemented with auxin and kinetin in the dark. Colored sections having deep yellow or orange color cells have been maintained for four years (32 generations) by subculturing on Linsmaier-Skoog Gelrite medium containing 1.0 mg/liter of indole-3-acetic acid and 0.1 mg/liter of kinetin in the dark. High-performance liquid chromatography of the yellow pigment showed the presence of crocin as a main component, but at low levels. An excess of gentiobiose, which was tentatively identified by high-performance liquid chromatography, was liberated from the yellow pigment by ammoniacal hydrolysis, but its origin other than crocin remains unknown.     

Growth and Morphogenesis in Tissue Cultures of Anagallis arvensis

Morphogenesis has been induced in excised organs and callus tissue cultures obtained from various parts of the seedling and mature plants of pimpernel (Anagallis arvensis). Vigorously growing cell cultures capable of being periodically subcultured have been established in liquid as well as on the agar-solidified Murashige and Skoog's medium supplemented with 2,4-D (0.1 mg/1) + kinetin (0.1 mg/l) + coconut milk (10%). The callus tissue obtained from excised hypocotyl segments is white, soft, friable and fast growing, and has been subcultured over a period of two years without showing any sign of decline in growth. The optimum conditions for growth are at pH 5.9, temperature 27°C, and with 4% sucrose as the carbon source. Under appropriate nutritional supply these cultures can be manipulated to induce rhizogenesis in the suspension cultures, and buds and “embryo-like” structures on agar-solidified media. The excised leaves, hypocotyl and stem segments regenerate buds. Of the cytokinins used, 6-(y,y-dimethylallylamino)-purine proved to be the best for the number of cultures producing buds, as well as for the number of buds per culture. Anatomical studies revealed that buds arise from the epidermal and subepidermal layers of leaves and hypocotyl; these buds form shoots which eventually develop into plantlets.     

Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Effects of cytokinins on the auxin requirement and auxin content of tobacco calluses

High concentrations (0.1–1 mg/liter) of kinetin permittedcontinuous growth of auxin-requiring and cytokinin-nonrequiringtobacco calluses on a medium without auxin. This effect of kinetindid not seem to be due to perpetuating change in the tissuecharacter, because tissue was auxin-requiring when returnedto a kinetin free medium. Cytokinins, i.e. benzylaminopurineand geranylaminopurine, showed the same effect as kinetin inmaking auxin-requiring calluses grow without a supply of auxin. In auxin-requiring and cytokinin-nonrequiring calluses subculturedfor 3 years on a medium containing 1 mg/liter kinetin withoutauxin, at least 3 auxins were detected by bioassay; 2 in theacidic and 1 in the neutral fraction. One was identified asIAA by paper chromatography (bioassay), thin-layer chromatographyand gas chromatography. Reduced or no auxin activity was foundin calluses transferred to a medium without kinetin. Kinetinwas apparently required to maintain the endogenous auxin levelin callus tissues. Kinetin may act on the auxin requirement of callus via its effectson auxin metabolism. ¹ Part XVI in the series "Studies on Plant Tissue Cultures". (Received April 11, 1972; )     

In vitro morphogenesis ofCrambe maritima L.

Summary Tissue cultures isolated from the root ofCrambe maritima have now undergone 17 transfers over two years on a medium containing 10% coconut milk, 2.0 mg/l IAA + 0.8 mg/l kinetin. The cultures consist predominantly of organized structures varying in complexity from nodular to root-like outgrowths and teratomatous leafy shoots. Embryoid-like structures also occur.     

The Effect of Kinetin on the Occurrence of Acid Auxin in Coleus blumei

Excised shoots and potted plants of Coleus blumei Benth, were subjected to different treatments with kinetin solutions. Control treatments were made with water. Free and bound auxin were extracted with ether. The acid fractions were purified by electrophoresis to remove all traces of kinetin, and were then analyzed with the Avena straight-growth test. As compared with the controls, kinetin treatment increases the bound auxin, whereas the corresponding free auxin is unchanged. Also methanol extracts of treated stems contain more acid auxin than corresponding extracts of water-treated stems. This indicates that on methanol extraction not only free auxin but also some bound auxin is obtained. The extracted auxin behaves like indoleacetic acid in paper chromatography with four different solvent systems, as well as in gel filtration through Sephadex. New formation or decreased breakdown of auxin seem the most likely explanations for the observed effects of kinetin.     

Fatty acids in callus cultures: Influence of growth factors on fatty acid composition of total lipids in callus cells

Callus tissues were derived from cotyledon segments of Cucumis melo and Cucumis sativus. Four combinations of growth factors, i.e., naphthalene acetic acid (NAA) plus coconut water (CW); NAA plus kinetin; NAA plus 6-benzylaminopurine (BAP) and indole 3-butyric acid plus BAP, were incorporated in the medium for callus initiation as well as for growth of excised callus in culture for six passages. The proportion of total saturated to unsaturated acids and the ratio of linoleic to linolenic acid was influenced by the change in the type of auxin and cytokinin in the combinations used. A many fold increase of myristic acid was recorded for the indole 3-butyric acid plus BAP combination.     

Morphogenesis in Stem-callus Cultures of Convolvulus arvensis L

Convolvulus arvensis stem explants form callus when inoculatedon to 0.5 mgm/1 2, 4-D, 15 per cent coconut milk, and on to1 mgm/1 kinetin, 15 per cent coconut milk. In the latter casenumerous shoots are formed on the callus. Callus formed on explantson 0.05 mgm/1 2, 4-D, 15 per cent coconut milk formed shootswith in-creasing vigour when transferred to a medium (1) inwhich the 2, 4–D concentration is lowered by one-tenth,(2) which contains no auxin but kinetin at 1 mgm/1, (3) whichcontains kinetin at 1 mgm/1 and 15 per cent coconut milk. OnNAA/ kinetin media shoots may form in which the intenode developmentis suppressed, giving compressed shoots. The capacity of callusto form shoots may be retained or lost through repeated sub-culture.Roots are formed erratically. The results are discussed, particularlywith regard to the loss of morphogenetic potential and whetherthis is reversible.     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Auxin activity of substituted benzoic acids and their effect on polar auxin transport

Six dichloro-, 3 trichloro-, 2 triiodo-, and 3 heterosubstituted benzoic acids (amiben, dinoben, dicamba), and N-1-naphthylphthalamic acid have been tested for effects on growth and on polar auxin transport. Growth activity with and without kinetin was measured by effects on fresh and dry weights of 30-day cultures of fresh tobacco pith. Transport inhibition was measured by following uptake and output of IAA-2-¹⁴C through 10 mm bean epicotyl sections. The distribution of callus growth on vascularized tobacco stem segments was also observed. Avena first internode extension assays established the relative activities: dicamba > amiben > dinoben suggested by pith growth results. Growth effects of active compounds were similar with and without kinetin, except that amiben was less active with kinetin, while 2,3,6-trichlorobenzoic acid was more active with kinetin than alone. The weak auxin activity of NPA was confirmed. Transport experiments showed that NPA was the most inhibitory compound tested, followed by TIBA. Other compounds tested were at least 300 times less inhibitory to IAA transport. The best growth promoters were the least inhibitory to transport, and the most effective transport inhibitors were at best poor auxins. It is suggested that the weak auxin and auxin synergistic activity of TIBA (and perhaps 2,3-dichlorobenzoic acid) in extension growth tests arises from its inhibition of transport of endogenous or added auxin out of the sections, rather than from its intrinsic auxin activity. Chemically induced apolar callus growth on vascularized tobacco stem explants can arise from inhibition of native auxin transport, apolar growth stimulation by auxinic action of the test compound, or both.     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.