Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

Somatic embryogenesis from callus cultures ofNardostachys jatamansi

Callus cultures ofNardostachys jatamansi DC, an endangered medicinal and aromatic plant, were established using petiole explants on MS medium supplemented with 16.1 µM -naphthaleneacetic acid and 1.16 µM kinetin. Embryogenesis in these callus cultures took place only upon sequential subculture of the callus on media having gradually decreasing auxin (16.1 to 1.34 µM NAA) and simultaneously increasing cytokinin (1.16 to 9.30 µM kinetin) concentrations over a period of 7 months. Somatic embryo to plantlet conversion took place on a medium containing 9.30 µM kinetin and 1.34 µM NAA.     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

The effects of cold-pretreatment,auxins and carbon source on anther culture of rice

The effects of a cold pretreatment, the concentration of different auxins (2,4-D, NAA and IAA) and the type of carbon source (maltose and sucrose) on the induction of callus from anthers of three parental lines and four rice F₁ hybrids (Japonica × Indica, Indica × Japonica) were studied. The results indicated that a cold pretreatment was essential for the induction of callus from anthers of the parental lines and the F₁ hybrids. These effects were genotype dependent. Auxins were essential for the induction of callus, and the type and concentration of auxins also influenced this process, as well as the type of carbon source. The greatest induction of callus was by the hybrid Morelos A92 × Koshihikari after a cold pretreatment of 8 days using 10.74 M –napthaleneacetic acid and 30 g l^–1 maltose.     

In vitro organogenesis from shoot tip,internode, and leaf explants of Ulmus x ‘Pioneer’

Shoot tip explants of the hybrid cultivar Pioneer responded poorly to initial attempts to establish shoot proliferating cultures on Murashige and Skoog (MS) medium containing 2 or 4 µM benzyladenine (BA) with a four week subculture interval. A combination of weekly subcultures and an MS medium containing 2 µM BA produced shoot proliferating cultures sufficient for micropropagation. Shoot organogenesis was obtained when callus derived from internodes of actively elongating shoots was transferred from a primary medium containing various cytokinins to a secondary medium containing MS salts and 10 µM BA. These small shoots elongated when transferred to a medium containing 2.5 µM BA. Adventitious shoots also differentiated on leaf tissue of Pioneer elm. These shoots appeared to differentiate with little if any intervening callus from the margins of leaves of in vitro grown shoots where these leaves touched the medium (MS medium containing 2 µM BA). Tissue cultured shoots from all sources were rooted, acclimated, and transplanted to the greenhouse or field with good success.Salaries and research aupport provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and The Nursery Crops Research Laboratory. Journal Article No. 23-86.     

Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA)

Embryogenic callus was established using immature male flower of Musa acuminata cv. Mas. After 5–6 months of culture, embryogenic callus was obtained at 21.75±11.9 from 750 immature male flower clusters with translucent somatic embryos proliferated from the whitish friable callus. It was observed that flower clusters ranging from 4 to 11 responded to form embryogenic callus and out of which 3–10 somatic embryos were formed per flower cluster. Embryogenic callus were obtained at a percentage of 10.00±0.3 on M1 medium initially supplemented with 18 M 2,4-dichlorophenoxyacetic acid (2,4-D) for 3 months and subsequently transferred to the same media with reduced 2,4-D (9 M) for the next 2–3 months. Embryos developed into translucent spheres and slightly torpedo shaped embryos in suspension cultures. Plantlets were obtained on medium M4 supplemented with 0.8M BA, at an average regeneration rate of 13.00±0.58.     

Callus production,suspension culture and in vitro alkaloid yields of Ephedra

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70±7h were recorded for cell suspension cultures of E. andina grown in a semi-continous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads.Neither parent plants or in vitro cultures of E. distachya, E. fragilis or E. saxatilis produced alkaloids. Trace quantities of 1-ephedrine and trace-0.14% dwt d-pseudoephedrine were produced by in vitro cultures of other species. The ability to produce alkaloid diminished to zero with successive subcultures.Abbreviations Eph 1-ephedrine - Peph d-pseudoephedrine - RGR relative growth rate - KIN kinetin - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA 3-in-dolebutyric acid - IAA indole-3-acetic acid     

Effects of the growth regulator 4PU-30 on growth,K+ content,and alkaloid production in tobacco callus cultures

Growth, K⁺ content, and alkaloid production were compared in nonorganogenetic callus cultures ofNicotiana tabacum cv. Burley 21 grown at 25°C in the dark on two different media: a basal medium with 1 M -naphthaleneacetic acid and 1 M kinetin, and one with 1 M -naphthaleneacetic acid and 1 M 4PU-30 (N-(2-chloro-4-pyridyl)-N-phenylurea). These callus tissues behaved differently not only in growth and K⁺ content but also in alkaloid production. In comparison to cultures grown with kinetin, those grown with 4PU-30 showed a significantly higher fresh weight and dry weight and K⁺ content during the growth period studied. The data clearly indicate a positive correlation between K⁺ uptake rate stimulated by 4PU-30 and cell enlargement rate. However, the alkaloid biosynthesis in the callus tissues was activated by the supply of kinetin and diminished by that of 4PU-30. It thus appears that cellular enlargement of meristematic tissue stimulated by 4PU-30 limited alkaloid production.     

Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graecum than by the differentiated plant

Callus cultures of Trigonella foenum-graecum contained 3 to 4 times more trigonelline than the seeds of this plant and 12 to 13 times more than the roots and shoots. Even higher levels of this alkaloid were produced by suspension cultures. This high productivity was maintained during successive subculturing of calli and cell suspensions for eight months. Thus, trigonelline is to be added to the group of the few metabolites whose synthesis in cell cultures exceeds its production in the differentiated plants. Media that had supported the growth of suspension cultures contained one third or more of the total alkaloid, whereas media of callus cultures contained about one tenth of this substance. Trigonelline accumulated in callus and suspension cultures with aging. Raising the level of nicotinic acid in the nutrient medium resulted in some increase of trigonelline production by the culture.Abbreviations 2.4 D 2.4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - GA Gibberellic acid - K kinetin     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkloids and anthraquinones

The influence of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth and the production of alkaloids and anthraquinones in cell suspension cultures of Cinchona ledgeriana Moens was studied. It was found that maximum growth and maximum alkaloid yield were obtained with a B₅-medium containing the normal level of nitrate and 4% sucrose, whereas the anthraquinone yield was maximum at 8% sucrose at the normal level of nitrate. Maximum contents of secondary metabolites, expressed as g.gDW^-1, were found using a medium containing 2% sucrose, but four times the normal nitrate concentration.     

Micropropagation of Alnus cordata (Loisel.) Loisel.

Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4 M 6-benzyladenine and 87.6 mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 M 6-benzyladenine and 333 mM glucose (multiplication medium) for a further 20–25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1–4.4 M 6-benzyladenine and 333 mM glucose 4–5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.2–4.9 M indolebutyric acid. Also, 98% rooting was achieved when cultures were treated with 625 mgl^-1 indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.     

Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.     

Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.)

Improved suspension cell culture systems are needed to facilitate the application of recombinant DNA technology for wheat germplasm enhancement. This study evaluated three wheat (Triticum aestivum L.) cultivars, and the effects of medium basal salts, 2,4-D, sucrose, and L-proline concentrations on the establishment of rapidly growing and highly embryogenic callus and suspension cultures. Percent embryogenic calli was visually estimated and verified with light and scanning electron microscopy. The most highly embryogenic callus was produced by cultivar Bobwhite on medium with MS basal salts, 5.6 M 2,4-D, 58 mM sucrose, and zero proline. The suspension cultures that produced the greatest number of regenerated plants utilized callus tissue produced on solid medium with MS basal salts, 87 mM sucrose, 9 M 2,4-D, and no proline.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid; RG, relative growth - %EC percent embryogenic calli - RV Redway and Vasil medium (1990a) - DPA days postanthesis     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

Growth and alkaloid production of callus culture induced from Securinega suffruticosa

The growth of, and production of alkaloids by, callus derived from budding stem explants of the germinated seeds of Securinega suffruticosa has been studied. The major alkaloids produced were securinine and allosecurinine with the latter being present in the greatest amount. The effects of pH, growth hormones, sucrose concentration and light and dark on callus growth and alkaloid production have been examined in detail. The pattern of alkaloid production in the callus culture appeared to be similar to that in the root of the securinega plant.     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone