Showing papers on "Kinetin published in 1999"

Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell

Abstract: Adventitious shoot buds were induced from leaf and stem explants of Bacopa monnieri on Murashige and Skoog medium supplemented with benzyladenine or kinetin. The source of the explants as well as different gelling agents in the medium were found to influence shoot induction and eventual shoot growth. The best response was obtained in leaf explants taken from shoot cultures grown in medium supplemented with 2 μM benzyladenine and gelled with 0.2% gelrite. A transverse section of the leaf explant incubated in this medium showed several shoot primordia emerging from the leaf surface. This system exhibited a potential for repeated harvesting of the shoots from the original leaf explant as the latter continued to expand and regenerate new shoots, upon repeated periodical subculturing onto fresh medium. However, the callusing response of the plant was very low. Qualitative TLC studies of the regenerated shoots revealed a phytochemical profile similar to that of the field grown-plants.

Factors regulating ethylene biosynthesis in etiolated Arabidopsis thaliana seedlings

Abstract: ethylene in other plant tissues. Auxin, cytokinin, brassinosteroid and cupric ion were found to highly elevate ethylene show that the synergism between cytokinin and auxin in production in these seedlings, but several other signaling com- Arabidopsis is due to an enhancement of the effects of auxin, pounds, as well as wounding and mechanical stimulation, had but not by increased elevation of ACS4 mRNA levels. These little or no effect. A mutant that disrupts the ACS5 gene results suggest that cytokinin acts post-transcriptionally to (cin5) was partially defective in the induction of ethylene in increase ACS4 function, which, coupled with the observation that auxin elevates ACS4 mRNA levels, accounts for the the presence of brassinosteroids, suggesting a role for this synergistic interaction. isoform in mediating this response. Cytokinin displayed a synergistic interaction with both brassinosteroid and auxin,

Somatic embryogenesis from mature leaves of rose (Rosa sp.)

Abstract: Several plant growth regulators (0.3–53.3 μm 6-benzyladenine, 2,4-dichlorophenoxyacetic acid, gibberellic acid, 3-indoleacetic acid, p-chlorophenoxyacetic acid, kinetin and α-naphthylacetic acid), alone or in combination, and culture conditions were tested for their capacity to induce somatic embryogenesis from mature leaf and stem explants of rose (Rosa sp.) of four commercial rose cultivars (Baccara, Mercedes, Ronto and Soraya). Somatic embryos were only induced from mature leaf explants derived from Soraya on Murashige and Skoog (MS) medium supplemented with 53.5 μm p-chlorophenoxyacetic acid and 4.6 μm kinetin, although satisfactory callus induction rates were obtained from all cultivars. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto a MS medium supplemented with 5.2 μm 6-benzyladenine and 5.7 μm 3-indoleacetic acid. Germination of mature embryos took place after subculturing them onto medium of the same composition. Plantlets regenerated from embryos and bearing three to four leaves were transferred to a greenhouse.

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

Abstract: A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10–5 m), kinetin (Kn) (5×10–6 m), 1-indolebutyric acid (IBA) (2×10–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l–1), or MS with 2,4-D (1×10–5 m), 6-benzylaminopurine (BAP) (1×10–5 m), and soluble PVP (250 mg l–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10–6 m), Kn (5×10–6 m) and soluble PVP (250 mg l–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10–6 m) and IBA (2.5×10–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced.

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

Abstract: The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m–2 s–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively.

Effects of kinetin on growth, grain yield and some mineral elements in wheat plants growing under excess salinity and oxygen deficiency

Abstract: Wheat plants, 22d. old, were exposed to wide range of soil water osmotic potential (Ψs = 0 to −1.2 MPa) induced by NaCl and CaCl2 treatments in combination with roots maintained under aerobic (drained at field capacity) or nonaerobic (flooded) conditions in the soil, and sprayed with 10 mg L−1 kinetin solution. In drained plants, not receiving kinetin, increased soil salinity resulted in appreciable inhibition of shoot growth and reduction in chlorophyll (Ch1.), soluble sugars (SS) contents and grain yield. Shoot growth, Ch1. content, soluble sugars and grain yield were significantly lower for flooded plants than unflooded analogues over the entire Ψs range. Both salinity and waterlogging synergize to increase Na+, Ca+ and Cl− accumulation in shoot tissues and to decrease the stability of leaf membranes to either dehydration (40% polyethylene glycol 6000) or heat (51 °C) stress. The ratio of K+/Na+ transported to shoots under aerobic and anaerobic conditions decreased progressively on salinization. The association between the internal mineral element concentrations was largely affected by kinetin treatment. Kinetin application ameliorated the deleterious effects of salinity and oxygen deficiency. It reduced Na+, Ca2+ and Cl− accumulation and improved K+ uptake under salinity and waterlogging stresses. Increased K+/Na+ ratio helped the plants to avoid Na+ toxicity and enhanced shoot growth and grain yield. Kinetin also reduced membrane injury by dehydration and heat stresses and improved the water status of plants under both aerobic and anaerobic conditions. The effects of single factors (Soil salinity ‘Ψs’, soil waterlogging ‘WL’ and Kinetin ‘Kin’) and their interactions (Ψs × WL, Ψs × Kin, WL × Kin and Ψs × WL × Kin) were shown by analysis of variance to be statistically significant for most parameters tested. Calculation of the coefficient of determination (η+) led to three important findings. (1) Salinity (Ψs) was dominant in affecting leaf relative water content (RWC), shoot dry mass, grain yield, stability of leaf membranes to dehydration stress and the contents of Na+, Ca2+, Mg2+ and Cl−. (2) Kinetin (Kin) had a dominant effect on the stability of leaf membranes to heat stress as well as on chlorophyll and soluble sugars contents. (3) The share of waterlogging (WL) was dominant for K+ content. It can be concluded that kinetin application helped wheat plants to grow successfully in the areas subjected to combined effects of salinity and oxygen deficiency, such as in salt marshes.

In vitro regeneration of a mature leguminous liana (Bauhinia vahlii Wight & Arnott)

Abstract: An in vitro propagation protocol has been developed from mature lianas of Bauhinia valii. Browning was the major obstacle in the establishment of cultures. Explants collected during the growing season (April–June) showed maximum browning; however, browning was minimal during the dormant phase. This problem was circumvented by soaking the sterilized explants in a solution of antioxidant (50 mg l–1 ascorbic acid+75 mg l–1 citric acid). The explants were thereafter transferred to culture room conditions after an initial incubation in the dark at 4 °C for 48 h. Shoot proliferation (58%), shoot number (4.5) and shoot length (35 mm) was best in Murashige and Skoog (MS) medium supplemented with 2.5 μM kinetin + 100 mg l–1 adenine sulfate. Seasonal fluctuations significantly affected the proliferation potential of the explants. March– April was found to be the best season for shoot initiation. Microshoots were rooted on a half-strength, growth regulator-free, agar-gelled Murashige and Skoog medium after a dip in half-strength MS liquid medium containing 1-naph-thaleneacetic acid + indole-3-butyric acid (10 μM). Rooted plantlets were potted and acclimatized under culture room conditions for 4 weeks before transfer to a polyhouse.

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Abstract: Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions.

Promotive Effects of the Peptidyl Plant Growth Factor, Phytosulfokine-α, on the Growth and Chlorophyll Content of Arabidopsis Seedlings under High Night-time Temperature Conditions

Abstract: In order to investigate the function of the peptidyl plant growth factor, phytosulfokine-alpha (PSK-alpha), in plants, we examined the effect of PSK-alpha on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions. Although exposure to high night-time temperatures markedly reduced the fresh weight and chlorophyll content of the seedlings, these parameters in the plants supplied with PSK-alpha remained at the same levels as those of non-treated controls. These effects were not apparent when [2-5]PSK, Tyr-SO3H and kinetin were similarly supplied. The results suggest that PSK-alpha not only promotes cell proliferation, but may aid plants in their tolerance of heat stress.

Intergeneric somatic hybrids of rice [Oryza sativa L. (+) Porteresia coarctata (Roxb.) Tateoka]

Abstract: Somatic hybrid plants were obtained following the electrofusion of rice (Oryza sativa L. cv ’Taipei 309’, 2n = 2x = 24) cell suspension–derived protoplasts with non-dividing leaf protoplasts of Porteresia coarctata (2n = 4x = 48), a saline-tolerant wild species. Fusion-treated protoplasts were plated on the surface of cellulose nitrate filter membranes, overlaying Lolium multiflorum nurse cells. The nurse cells were embedded in KPR medium containing 0.5 mg l−1 2,4–dichlorophenoxyacetic acid and semi-solidified with SeaPlaque agarose. Putative somatic hybrid cell colonies were selected on the basis of their growth, whereby faster growing colonies were transferred preferentially to MS-based medium with 2.0 mg l−1 kinetin, 0.5 mg l−1α-naphthaleneacetic acid, 30 g l−1 sucrose and 4.0 g l−1 SeaKem agarose to induce shoot regeneration. One hundred and nineteen regenerated plants were micropropagated clonally on MS-based medium containing 2.0 mg l−1 6–benzylaminopurine, 50 g l−1 sucrose and 4.0 g l−1 SeaKem agarose, prior to DNA extraction of plant samples. Putative somatic hybrids were initially identified by RAPD analysis, and 8 plant lines were selected for further investigation by flow cytometric ploidy determination and cytology. Plants of one line had an allohexaploid chromosome complement (2n = 6x = 72) and, following examination of its vegetative clones by GISH, were confirmed as somatic hybrids containing full chromosome complements of both O. sativa and P. coarctata.

Role of kinetin in alleviation of copper and zinc toxicity in Lupinus termis plants

Abstract: The effect of exogenous kinetin application on the growth and some physiological processes of Lupinus termis plants growing in metal containing solutions with excess concentrations of Cu and Zn ion were studied Generally, plants growing in these solutions had a lower chlorophyll (Chl) content, leaf relative water content (RWC) and produced less biomass than the control plants Proline content was higher in metal-treated plants than in untreated controls Chromatography of cell-free-extracts of roots and shoots indicated three main protein peaks with molecular weights about 170, 75--70 and 5--45 kDa These peaks were coincident with Cu or Zn maxima Addition of kinetin reduced the decline in Chl content in metal-treated plants, improved water status of the plants and enhanced growth of the shoots and roots The Cu or Zn content expressed on a per mg protein basis was raised when kinetin was applied to the growing shoots Kinetin (Kin), Cu and Zn, singly and in the presence of kinetin (Cu × Kin and Zn × Kin), significantly affected the parameters tested Only the effects of Cu × Kin and Zn × Kin interactions on shoot fresh weight and Cu × Kin on root length were statistically insignificant Based on the calculated coefficient of determination (η 2) the roles of Cu and Zn in affecting Chl content and growth were dominant in comparison to kinetin Kinetin effect was dominant for root length and proline content, but the role of the interaction was subdominant The results of this study indicate that kinetin can alleviate the harmful effects of Cu and Zn on the growth of lupin plants through stimulation of Cu and Zn incorporation into metal-binding proteins

Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomile ( Chamomilla recutita L.)

Abstract: A protocol has been developed for the induction of somatic embryogenesis from flower explants of chamomile (Chamomilla recutita L.). The effects of several plant growth regulators [α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA) and kinetin (Kin), alone or in combination] and the flower type (disk or ray flower) were investigated. Both types of flowers responded to the callus and shoot induction treatments, but formation of globular somatic embryos took place only on disk-flower-derived explants after 2–4 weeks of culture on a Murashige and Skoog (MS) medium supplemented either with 8.87 μm BA and 1.07 μm NAA or with 26.8 μm NAA and 11.5 μm Kin. However, fully developed, cotyledonary-stage somatic embryos could be induced only on the NAA/Kin medium, 10 weeks after culture initiation. Germination of the embryos and plant regeneration took place after subculture for 4–5 weeks onto medium of the same composition. Plantlets regenerated from embryos flowered in vitro on a MS medium supplemented with 8.87 μm BA and 1.07 μm NAA. The significance of the results with respect to chamomile micropropagation and the utilization of wild populations in breeding programs is discussed.

Effects of lead and kinetin on the growth, and some physiological components of safflower

Abstract: Pollution of the root environment with excess of Pb retarded shoot growth, decreased chlorophyll (Chl) content and reduced Chl stability (CSI) to heat. Plants growing in Pb polluted soil accumulated much more free amino acids and less soluble sugars than the control plants. Stability of leaf membranes to heat (51 °C) or dehydration stresses (40% polyethylene glycol, 6000) decreased in response to Pb pollution where the membranes of leaf discs excised from Pb-treated plants were damaged more than those taken from plants growing in Pb free soil. Supplying kinetin ameliorated the deleterious effects of Pb pollution on the parameters tested. Kinetin-treated plants had higher Chl, soluble sugars content and produced more biomass in their shoots. Also, kinetin increased leaf membrane stability especially in Pb-treated plants, effectively protected chlorophyll degradation by heat and increased Chl a and b stability index; the most effective concentration was 10 mg L−1. The effects of Pb and kinetin as well as their interaction (Pb × Kin) on the parameters tested were statistically significant. Applied kinetin had a dominant role (as indicated by η2) in affecting shoot growth, soluble sugars, Chl a and b contents, stability of leaf membranes to dehydration stress as well as the Chl stability index. Pb had a dominant role on total free amino acids (TAA) and leaf relative water content (RWC). The interaction between Kin × Pb influenced the stability of leaf membranes to heat stress in a major way.

In vitro organogenesis of Polypodium cambricum

Abstract: Sporophyte regeneration through green globular bodies (GGB) is affected by the supply of growth regulators as well as by the source of the donor explant. Rhizome, frond, petiole or root tip explants from juvenile sporophytes of Polypodium cambricum were cultured on media containing N6-benzyladenine (BA) or Kinetin alone or in combination with α-naphthaleneacetic acid (NAA). GGB production took place even in growth regulator-free medium. Addition of BA into the culture medium was very effective to induce formation of GGB in rhizome explants. Root tip explants exhibited the lowest organogenic capacity. Homogenization of BA-pretreated frond or rhizome was a powerful system to improve fern propagation.

Somatic embryogenesis in Canary Island date palm

Abstract: Zygotic embryo and shoot tip explants of Phoenix canariensis were cultured on MS (1962) basal medium supplemented with 100 μM Picloram and 9.5 μM kinetin or 10.8 μM or 45.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.8 μM N6-(2-isopentenyl) adenine (2iP). These explants after 12 weeks in darkness at 28 °C, produced embryogenic callus with very compact, pale yellow, nodular structures. Proliferation and maintenance of embryogenic callus was on MS basal medium with 2.26 μM 2,4-D, 0.83 μM kinetin and 2 μM abscisic acid (ABA), with a regular subculture every 3–4 weeks. Somatic embryo development was promoted by two months of culture on MS liquid medium enriched with 2 μM ABA, for torpedo stage development, then on liquid MS medium with 1 μM N6-benzyladenine (BA) and 0.46 μM kinetin, for shoot induction. Germinated embryos were transferred to basal media enriched with 0.45 μM BA and 0.06 μM naphthaleneacetic acid (NAA) for root and shoot induction and elongation. Viable plants were recovered on basal MS free of plant growth regulators (PGRs), but percentages of plant conversion have to be improved.

In vitro propagation of a semi-dwarfing cherry rootstock

Abstract: A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media.

Efficient Plant Regeneration from Suspension-cultured Cells of Tall Bearded Iris

Abstract: A protocol was developed for efficient plant regeneration of Iris germanica L. 'Skating Party' from suspension cultures. Suspension cultures were maintained in Murashige and Skoog (MS) basal medium (pH 5.9) supplemented with 290 mg·L -1 proline, 50 g·L -1 sucrose, 5.0 μM 2,4-D, and 0.5 μM Kin. Suspension-cultured cells were transferred to a shoot induction medium (MS basal medium supplemented with 10 mg·L -1 pantothenic acid, 4.5 mg·L -1 nicotinic acid, 1.9 mg·L -1 thiamine, 250 mg·L -1 casein hydrolysate, 250 mg·L -1 proline, 50 g·L -1 sucrose, 2.0 g·L -1 Phytagel, 0.5 μM NAA, and 12.5 μM Kin). Cell clusters that proliferated on this medium differentiated and developed shoots and plant- lets in about 5 weeks. Regeneration apparently occurred via both somatic embryogenesis and shoot organogenesis. A series of experiments was conducted to optimize conditions during suspension culture to maximize subsequent plant regeneration. Parameters in- cluded 2,4-D and Kin concentrations, the subculture interval, and the size of cell clusters. The highest regeneration rate was achieved with cell clusters ≤280 μm in diameter, derived from suspension cultures grown for 6 weeks without subculturing in liquid medium containing 5 μM 2,4-D and 0.5 μM Kin. Up to 4000 plantlets with normal vegetative growth and morphology could be generated from 1 g of suspension-cultured cells in about 3-4 months. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); kinetin (Kin); 1- naphthaleneacetic acid (NAA). Iris germanica is one of the horticulturally most important tall bearded irises in the genus. Hundreds of valuable cultivars from this spe- cies have been developed and cultivated com- mercially as perennial ornamental plants. Tra- ditionally, rhizomatous iris plants are propa- gated by splitting rhizomes, with a maximum annual yield of 10 plants/rhizome (Jehan et al., 1994). This practice is inefficient and slow, especially for propagating new cultivars for commercial use. Propagation by seed is im- practical because of low germination rates and the allogamous nature of iris. Therefore, a more efficient propagation method is needed. Also, strong consumer demand increases the challenge of developing new cultivars with novel flower characteristics. Introducing de- sirable traits by genetic transformation would offer an attractive means for iris cultivar im- provement.

Production of the sedative triterpene galphimine B in Galphimia glauca tissue culture.

Abstract: A tissue culture method is described for callus formation from Galphimia glauca (Mapighiaceae) in MS (Murashige & Skoog) medium supplemented with various growth regulators. Best induction was achieved when using hypocotyls as explants in medium supplemented with 2 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D). Major cellular growth of calluses was obtained with naphthaleneacetic acid (2 mg/l) + kinetin (1 mg/l). Subcultivation of calluses using various concentrations of 2,4-D allowed the production of the sedative nor-seco-triterpenoid, galphimine B and a new related compound. The structure of the new constituent was elucidated as 6-acetoxygalphimine B. The highest accumulation of active constituent 1 (1.5 x 10(-2) dry weight) was achieved when 4 mg/l of the hormone were used, and this experimental condition allowed the detection of only galphimine B. A preliminary screening of the methanolic extracts prepared from calluses, using the isolated guinea-pig ileum as a general test system for pharmacological effects, demonstrated that the most active material was the one with the highest concentration of galphimine B. Total accumulation of this sedative triterpene, in the optimized tissue culture conditions, was in the same order of magnitude as the value quantified for wild plants (4.5 x 10(-2) dry weight).

Culture and regeneration of mesophyll-derived protoplasts of sorghum [Sorghum bicolor (L.) Moench]

Abstract: A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l–1 kinetin and 2 mg l–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity.

The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Abstract: Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10–6m 2,4-D, 10–6m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols.

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Abstract: Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 µM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 µM) kinetin per 1, 1 mg of both 2,4-D (4.5 µM) and kinetin (4.7 µM per 1, or 2 mg of both 2,4-D (9.1 µM) and kinetin (9.3 µM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 µM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.

Factors affecting somation embryogenesis in long term callus cultures of 'safed musli' (Chlorophytum borivilianum), an endangered wonder herb

Abstract: ChiorophYlU lIl boriniianu7Il, commonly known as ' safed musli' , has become an endangered species due to ,t s ,,:cr-exploitation for tuberous roots, used as tonic and aphrodisiac. An improved method has been ut ,' cl oped fo r large-scale rapid multiplication of C. borivilianum through somatic embryogenesis in callus cu ltures. Somatic embryos were obtained on MS medium containing 2.25 J..lM 2,4-D and 1.15 ~lM kinetin . The concentration and com ination of KNO) and (NH4)2S04 in the medium had a marked effect on the somatic embryogenesis . Anunonium to nitrate nitrogen in the ratio of 1:4 at low total ni­ trogen level (250 or 500 mgNL" ) in th e medium was favourable for the growth and somatic embryo­ genesis. Among th e various cytokinins tested, 2-iP had a maximum stimulatory effect on somatic em­ bryogenesis. Decreasing order of the effectiveness of various cytokinins incorporated in MS-9 medium (i .e., MS medium containing 100 mgNL' 1 (NH4)2S0 4 and 400 mgNL' 1 KJ~03) supplemented with 2.25 J..lM 2,4-0 was 2-iP > TDZ >kinetin >BAP. Incorporation of amino acids, viz. glutamine and proline, promotes embryogenesis. Maximum number of somatic embryos (63 per inoculum) were observed on MS-9 medium supplemented with 100 mgL"' proline, 1. 12 J..lM 2,4-D and 7.42 J.1M 2-iP. Germination of somatic embryos, isolated from young cultures (1-5 months-old) was maximum (48%) on MS-9 me­ d ium supplemented with 17.6 J.1M BAP. On the other hand, somatic embryos developed in long-term cultures were abnormal and showed a wide range of variation in the number of chromosomes, ranging from 23 to 58 (i.e" 3X-3 to 7X+2) along with several structural chromosomal abnormalities. This may be the reason fo r the decline in the germination potential of somatic embryos in long-term cultures. Thus, callus produced from shoot cultures obtained from stem-disc explants on B5 medium containing 2 J..lM TDZ and 0 .18 mM adenine, provides an excellent source for obtaining fresh cytologically-stable embryogenic cultures. Plamlets developed in vitro through embryo germination were successfully trans­ ferre d to soil with 90% survivability . The technique developed is highly efficient to get miniature plantlets for fiel d transfer in 2 months, starting from the callus .· Chlorophytum borivilianum (liliaceae) and a few other species are commonly known as 'safed musli'. Tuberous roots of the plant are widely used as tonic and aphrodisiac due to the presence of steroidal saponins, viz . neotigogenin, neoheco­ geni n, sti gmasterol and tokorogenin I. 2. C. borivilianum has been used along with other plants such as Asparagus ascendense, A. racemosum, Curculigo orchioides and Withania somnifera in several formulations in the Indian system of medi­ ci ne 3 . 4 • Pharmocological investigations of saponin

Effect of growth medium composition on psilostachyinolides and altamisine production

Abstract: Callus cultures of Ambrosia tenuifolia were established from sub-apical leaves. The explants were grown on basal media MS (Murashige and Skoog) supplemented with 10 μM kinetin, 1 μM 2-4 dichlorophenoxyacetic, ascorbic acid and cysteine and cultivated either in light or darkness. To determine the effect of each individual salt component on the growing rate of the callus and the psilostachyinolides and altamisine production in the culture, the concentrations of each nutrient was tested at different levels, ranging from 50% above to 50% below the standard medium. Interestingly, increased concentration of boron in the medium, resulted in a four fold increase in the production of sesquiterpene lactones by the callus. However, positive production of psilostachynolides and altamisine was only detected when the basal concentrations of the others components of the basal media were used. In addition, production of altamisine was highly sensitive to the variations of nutrients. No statiscally effect on terpenoide production by the callus was detected by varying light exposure. These results indicates that conditions for the optimal production of these terpenoids can be enhanced by modifying nutrients of the MS basal media.

2,4-Dichlorophenoxyacetic acid and kinetin in anther culture of cultivated and wild oats and their interspecific crosses: plant regeneration from A. sativa L.

Abstract: The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin was studied in anther culture of oat Avena sativa L., wild oat A. sterilis L. and progeny of crosses between them. A high 2,4-D concentration (5–6 mg l–1) increased embryo production in genotypes of both species and promoted plant regeneration in anther cultures of A. sterilis and A. sativa×A. sterilis progeny, while kinetin caused severe browning. However, a low concentration of kinetin was essential for initiation of regenerable embryos from anther culture of A. sativa cv. Kolbu: one green and one albino plant were produced. In addition, medium containing W14 salts gave higher regenerant recovery compared with medium containing Murashge and Skoog salts, when cross progeny were tested.

Organogenic Plant Regeneration in Bioreactors

Abstract: Plant micropropagation is as yet encumbered by intensive manual manipulation and requires scaling-up and automation to provide low cost propagation (Aitken-Christie 1991). Scaling-up necessitates liquid media in bioreactors (Preil 1991; Takayama 1991), and measures must be taken to arrest shoot and leaf growth to prevent shoot malformation — hyperhydricity (Ziv 1991a). Plants developing in liquid cultures exhibit abnormal hyperhydrated leaves and cannot survive transplanting ex vitro (Ziv 1991a, Ziv 1994). Shoot morphogenesis in vitro can be controlled by culture condition which limit or prevent leaf expansion and shoot growth, while at the same time enhances proliferation. The formation of spherical organized structures, in which the shoots are reduced to buds or meristematic tissue was reported in several species. Protocorm-like bodies formed in liquid cultured gladiolus (Ziv 1989, 1990) and Brodiaea (Ilan et al. 1995). In woody species McCown et al. (1988) and Aitken-Christie et al. (1988) described nodular structures in poplar and meristematic tissue in radiata pine respectively. In bioreactor cultured fern shoots kinetin (KIN) and adenine sulphate induced the formation of meristematic clusters (Ziv and Hadar 1991). Levin et al. (1988) described a several fold biomass increase of organogenic clusters of several species. The clusters were separated, sorted in a controlled bioprocessor and dispensed to agar cultures for further plantlet development.

6-Benzylaminopurine: A Plant Derived Cytokinin Inducing Positive Inotropism by P2-Purinoceptors

Abstract: Positive inotropic effects induced by 6-benzylaminopurine (6-BAP), kinetin and zeatin were studied in rat atria. The potency order observed was 6-BAP > or = kinetin > zeatin. Suramin, a P2-purinoceptor antagonist, inhibited the positive effect of 6-BAP suggesting the involvement of P2-purinoceptors in the positive effect of this cytokinin. In order to elucidate this point, 6-BAP was used against R-PIA (a P1-purinoceptor agonist) and ATP and UTP (both P2-purinoceptor agonists). 6-BAP did not influence negative inotropism by R-PIA whereas both nucleotides were inhibited after pretreatment with the cytokinin. LY 83583, an inhibitor of cGMP production, reduced the inotropic effect by cytokinin whereas L-NAME, an inhibitor of the L-arginine/nitric oxide pathway, did not influence the effect induced by 6-BAP. Indomethacin, an inhibitor of cyclooxygenase, and neomycin, an inhibitor of phospholipase C, did not significantly modify positive inotropism by 6-BAP. Verapamil, an inhibitor of L-type calcium channels, did not change the positive effect of 6-BAP while TMB-8 and dantrolene, two inhibitors of intracellular calcium release, reduced the increase of contractile tension induced by cytokinin. Our data on rat atria suggest that 6-BAP causes a positive inotropism through activation of P2-purinoceptors, involving modification of cGMP and of intracellular calcium.