Showing papers on "Murashige and Skoog medium published in 2001"

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

Abstract: A mass in vitro propagation system for Bacopa monniera (L) Wettst (Scrophulariaceae), a medicinally important plant, has been developed A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (68 μM) and 6-benzyladenine (89 μM) proved superior to other treatments Optimum adventitious shoot buds induction occurred at 68 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation However, subculture of leaf explants on medium containing 22 μM benzyladenine yielded a higher number (1291) of adventitious shoot buds by the end of third subculture The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 49 μM IBA After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field

Induction of Highly Embryogenic Calli and Plant Regeneration in Upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) Cottons

Abstract: To accomplish our objective of broadening the number ofregenerable cotton lines, we developed a protocol capable of producing plants through somatic embryogenesis of diverse cotton species. Callus was initiated from hypocotyl and cotyledon explants on a callus initiation medium [CIM; modified MS with 1 mg L -1 kinetin and 2 mg L -1 naphthaleneacetic acid (NAA) 1. Friable embryogenic callus was periodically selected and transferred onto callus selection/maintenance medium (CS/MM) [modified MS with 0.1 mg L -1 kinetin and 0.5 mg L -1 NAA]. The selected callus was then transferred into a liquid embryo initiation medium (EIM) (modified MS medium in which NH 4 NO 3 was removed and KNO 3 amount doubled) followed by transfer to solid embryo maturation media EMMS 2 (0.5 mg L -1 NAA + 0.05 mg L -1 kinetin). The liquid step not only decreased the culturing time but also increased the number of embryos per gram of cultured tissue. Germinating somatic embryos were placed on MS medium with no hormones and plantlets were acclimatized before transfer to the greenhouse. Significant numbers of somatic embryos and their derived plantlets were obtained from a commercial cultivar of G. hirsutum, Deltapine 90 and G. barbadense accession GB-35B126 (PI-528306). The mean embryos per gram for Deltapine 90 on EMMS 2 were higher than those previously reported for Coker 312. Highly significant differences were found between the two genotypes for both embryo and plant production. To our knowledge, this is the first report of regeneration of G. barbadense through somatic embryogenesis.

Micropropagation of Cunila galioides, a popular medicinal plant of south Brazil

Abstract: Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 μM of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 μM of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.

Micropropagation of the endangered Aloe polyphylla

Abstract: A rapid propagation protocol was establish for thehighly endangered Aloe polyphylla (Schonland ex Pillans). Seed was germinated in vitro on Murashige & Skoog 1962 medium [6] with or without sucrose. Plantlets were cultured onmedium containing benzyladenine only, or a combinationof BA and NAA. After initial problems with browning,the explants rapidly formed axillary and adventitiousbuds. Maximal shoot formation was obtained on MSmedium containing 1.0 mg l−1 BA. Some shootsrooted spontaneously on MS medium, but the rootingpercentage was improved with a 0.5 mg l−1 IBAsupplement. Rooted plantlets were acclimatized togreenhouse conditions. The success of this projectindicates that micropropagation can be a useful toolin the conservation of this endangered species of thegenus Aloe.

Regenerated plants in tylophora indica (burm. f. merrill.)

Abstract: embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.0-3 mg 1-1; 0.0-13.56 [LM) and kinetin (Kn; 0.01 mg 1-1; 0.05 pIM). The best response for callus induction was obtained on MS medium containing 2 mg 1-1 (9.04 pM) 2,4-D and 0.01 mg 1-1 (0.05 pM) Kn. After two subcultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5-3 mgl-1; 2.22-13.32 KpM) and 2-isopentenyladenine (2ip; 0.53 mg 1-1; 2.46-14.76 iM) along with 0.01 mg 1-1 (0.05 lM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg 1-1 (9.84 KM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in tylophora indica (burm. f. merrill.)

Abstract: A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l−1; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l−1; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l−1 (9.04 μM) 2.4-D and 0.01 mg l−1 (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l−1; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l−1; 2.46–14.76 μM) along with 0.01 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l−1 (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.

Endogenous hormone levels in explants and in embryogenic and non-embryogenic cultures of carrot.

Abstract: Carrot (Daucus carota L. F1 hybrid Starca) excised hypocotyls were cultured on Murashige and Skoog medium with and without 2,4-dichlorophenoxy acetic acid (2,4-D) to determine the effect of this plant growth regulator on their further development and their endogenous hormone levels. Culture in the absence of 2,4-D stimulated root development at one end of the hypocotyl segments and increased the endogenous levels of free indole-3-acetic acid (IAA), zeatin/zeatin riboside and N6(Delta2-isopentenyl) adenine/N6(Delta2-isopentenyl) adenosine, as determined by radio-immunoassay. On the other hand, the presence of 2,4-D in the culture medium promoted callus induction and proliferation, together with abscisic acid (ABA) accumulation, in the hypocotyl segments during the first weeks of culture. When the callus segments generated in the hypocotyl sections cultured in the presence of 2,4-D were cultivated further, the development of two callus types was observed, one composed of preglobular and globular embryos and the other translucent, watery and lacking any sign of organisation. The embryos of the first type germinated when callus segments were transferred to regeneration conditions, while no change was observed when the second type was induced to regenerate. Higher levels of free IAA and ABA were obtained in the embryogenic calli when compared to the non-embryogenic, while no differences were observed among callus types in the other hormones evaluated. The possible role of the different plant hormones during induction of somatic embryogenesis is discussed.

Production of withaferin A in shoot cultures of Withania somnifera.

Abstract: Multiple shoot cultures of Withania somnifera were established from single shoot tip explants and their potential for the production of two principle withanolides, withaferin A and withanolide D was investigated. Shoot tips grown on MS medium supplemented with BA (1 mg l-1) induced 10.0 ± 1.15 microshoots per explants and shoot cultures accumulated both withanolides (withaferin A = 0.04 %, withanolide D = 0.06 %). Supplementation of MSSM (solid) agar medium with 4 % sucrose enhanced accumulation of both withaferin A (0.16 %) and withanolide D (0.08 %). Reduction of the agar concentration to 0.16 % increased the number of microshoots induced per explant to 25.5. MSSM liquid medium containing 10 % coconut milk favoured a maximum increase in biomass (27 fold); number of microshoots induced (37.6 ± 1.45) as well as accumulation of withaferin A (0.14 %). BA:N6-Benzyladenine Kn:Kinetin 2iP:N6-[2-Isopentenyl]adenine MS:Murashige and Skoog (1962) MSSM:Murashige and Skoog's basal medium + BA (1 mg l-1)

Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus

Abstract: A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54 μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than 150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant.

In vitro regeneration of Acacia mangium via organogenesis

Abstract: Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house.

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

Abstract: An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stemstTCLs were cut transversely along young stems from which the shoot-tipshad been removed Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl−1 agar, different sucrose concentrations (10, 20, 30 or 40g l−1), and with or without 1 mg l−1 activatedcharcoal (AC) Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l−1 agar and 20 gl−1 sucrose Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l−1 after 60days of culture No root formation occurred Both shooting and rootingoccurred when sucrose was used at 20 g l−1 The plantletswere transferred to soil and grew well under greenhouseconditions

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Abstract: Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.

Micropropagation of Rollinia mucosa (JACQ.) baill

Abstract: A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l−1 polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l−1 sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.

In Vitro Culture of Lingonberry (Vaccinium vitis-idaea L.)

Abstract: Shoots of three lingonberry (Vaccinium vitis-idaea L.) cultivars, ‘Regal’, ‘Splendor’, and ‘Erntedank’ and two clones from natural stands in Newfoundland were initiated in vitro from shoot tip and nodal explants on modified Murashige and Skoog (MS) medium containing the plant growth regulators N6-[2-isopentenyl]adenine (2iP) (12.3 μM) or zeatin (5.7 μM). Zeatin was more effective than 2iP, and induced proliferation of 2 to 3 times as many shoots in ‘Regal’ as 2iP. Out of four media tested for shoot proliferation, modified MS medium was found more effective than the Woody Plant Medium for shoot multiplication. With all three cultivars, a reasonable balance in terms of shoot multiplication rate and desirable growth characteristics was attained in a new medium. Nodal explants of the two clones cultured on the modified MS medium with 12.3 μM 2iP produced 4 to 6 healthy axillary shoots per explant in clone 1 and 2 to 4 shoots in clone 2. Shoots rooted well ex vitro directly on a 2 peat: 1 perlite (v/v...

Somatic embryogenesis from leaf cultures of potato

Abstract: An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and α-naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA3) and BA. Treatment with zeatin (22.8 μM) and BA (10.0 μM) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.

Regeneration of Acacia mangium through somatic embryogenesis

Abstract: Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA3 followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species.

Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro

Abstract: Continuous axillary shoot proliferation and in vitro flowering were achieved using single node explants from a mature (over 70-yr-old) field clump of Dendrocalamus giganteus (giant bamboo). The shoots proliferated in a basal Murashige and Skoog medium with 6 mgl−1 (26.6 μM) N6-benzyladenine (BA) and 2% sucrose. The rate of shoot proliferation gradually increased to over three-fold before in vitro flowering took place. In vitro flowering was not the expression of a species-specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower. The rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation leading to build up, and release of stress in relation to flowering and its reversion are discussed.

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Abstract: Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota.

Abstract: Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l–1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.

Effect of genotype, explant type and growth regulators on organogenesis in Morus alba

Abstract: Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l−1 2,4-D and 0.5 mg l−1 BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l−1 BA. Supplementation with 0.1 mg l−1 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l−1 indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well.

In vitro flowering of bitter melon.

Abstract: Flowers were formed from shoot tips of bitter melon (Momordica charantia L.) cultured on Murashige and Skoog medium supplemented with 90 mM sucrose, 0.05 mM Fe2+ and 4 µM N6-benzyladenine (BA). The addition of 0.05 mM Fe2+ to the medium prevented chlorosis of the explant and promoted normal flowering. Increasing the ratio of carbon to nitrogen promoted male flower formation but intensively inhibited vegetative growth. The influence of cytokinin on the morphogenesis of the explant was highly notable. Flowers could be formed after a 15- to 20-day exposure to kinetin (Kin) or BA. Kin and BA had opposite effects with regard to the development of the explant. Kin promoted flower formation, especially female, but inhibited branch bud formation. Conversely, BA promoted branch bud formation and also promoted male flower formation when present at a concentration of 1–2 µM, but completely inhibited flower formation at 4–8 µM. Fluorescein diacetate staining and in vitro germination showed that in vitro pollen were of a fairly high viability.

Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin

Abstract: Excised tissues from transverse young stem sections of Lilium longiflorum were cultured on Murashige and Skoog medium supplemented with growth regulators at various concentrations. After 45 d in culture, the presence of α-naphthaleneacetic acid (NAA) in the culture medium at 5.4 μM resulted in bulblet formation while 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.2 μM resulted in root formation. The presence of IBA (indole-3-butyric acid) in the culture medium at 1.0 μM resulted in shoot formation while plantlet formation occurred when IBA was added at a concentration of 2.0 μM. When 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) was added to the culture medium at 1.1 μM, protocorm-like bodies (PLBs) formed, while 2.2 μM resulted in shoot formation (on abaxial and adaxial surfaces). The presence of NAA and TDZ in the culture medium at 5.4 μM and 0.4, 1.1 or 2.2 μM, respectively, resulted in somatic embryo formation while NAA- and 6-benzylaminopurine-(BA) containing culture medium formed callus or bulblets. The establishment of different regeneration systems when explants are exposed to various growth regulators demonstrates that the choice of growth regulator combinations and concentrations are of significance in determining the morphogenetic response and plant regeneration capacity.

In Vitro conservation of enset under slow growth conditions

Abstract: Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage.

In vitro propagation and rhizome formation in Curcuma longa Linn.

Abstract: Turmeric (Curcuma longa Linn.) which is cultivated by underground rhizomes is a slow propagating species. Multiplication and callus induction starting from the rhizome buds and shoot tips of C. longa in MS medium was carried out. A combination of naphthalene acetic acid (NAA; 1.0 mg/l) with kinetin (Kn; 1.0 mg/l) or NAA (1.0 mg/l) with 6-benzylaminopurine (BAP; 2.0 mg/l) was optimum for rapid clonal propagation of turmeric. A concentration of 2.5-3.0 mg/l of 2,4-dichlorophenoxy-acetic acid (2,4-D) was found to be optimum for callus induction. Regeneration of plantlets from a callus was successfully conducted in MS medium supplemented with standard growth hormones for multiplication at 25 +/- 2 degrees C under a 16 h photoperiod. These plantlets were successfully transferred to the field. Plantlets (4-month-old) were incubated in a medium containing different concentrations of sucrose supplemented with NAA (0.1 mg/l) and Kn (1.0 mg/l) at 27 +/- 2 degrees C under an 8 h photoperiod for induction of rhizomes. In vitro rhizome formation was observed in media containing 6 and 8% sucrose.

Transformation of azuki bean by Agrobacterium tumefaciens

Abstract: Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the β-glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 μM acetosyringone and 10 mg l−1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l−1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l−1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.

Establishment and analysis of fast-growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant

Abstract: Fast-growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant was established from leaf and inter nodal explants of in vitro-raised shoot cultures in Murashige and Skoog (MS) medium containing 2.5 mg/l 6-benzyladenine (BA), 0.5 mg/l 2-isopentenyladenine (2-ip) and 0.5 mg/l α-naphthaleneacetic acid (NAA). Shoot cultures were maintained on MS agar medium supplemented with 0.5 mg/l BA and 0.05 mg/l 2-ip or and 0.05 mg/l NAA and subcultured at 5 -weeks interval. Leaf explants incubated in total darkness in half strength MS medium supplemented with 0.5 mg/l IBA and 0.2 mg/l NAA favoured induction of roots in 89% of the cultures with highest biomass of roots (5.820 g). A 100 mg fresh root tissue cultured in 80 ml half -strength MS liquid medium supplemented with 0.2 mg/l IBA and 0.1 mg/l NAA, under continuous agitation (80 rpm), yielded 3.433 g and 0.734 g fresh and dry weight of roots, respectively. Roots grown in this optimal medium produced maximum compound, 2-hydroxy-4-methoxy benzaldehyde (0.16%) after 6 weeks of culture. The root cultures were maintained up to the 7th passage without decline of growth. DECALEPIS arayalpathra (Joseph & Chandras) Venter., (Janakia arayalpathra Joseph & Chandras) (Periplocaceae) is a perennial woody laticiferous shrub 1,2 with slender, spreading branches. The plant is endemic to southern forests of the Western Ghats region of Kerala (South India) 3 , distributed at an elevation of 800 – 1200 m and growing in the crevices of rocks. The monoliform tuberous roots of the plant are highly ar omatic and the native Kani tribes use it as an effective remedy for peptic ulcer, cancer-like afflictions and as a rejuvenating tonic 4 . Recent pharmacological investigations of the root extract of the plant reveale d immun omodulatory and anticancer properties 5 . The tubers are being ruthlessly collected from its natural habitat by the local Kani tribes. This has led to the acute scarcity of the plant. Consequently, it has been enlisted as enda ngered plant species 6 . The natural regeneration as well as conventional propagation of this plant is beset with se veral factors like poor fruit set, seed germination and rooting on stem cuttings. Considering the urgent need

Factors Affecting In Vitro Multiplication of Date Palm

Abstract: Rapid method of in vitro multiplication of date palm was developed Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm−3 dimethylaminopurine (2iP) + 1 mg dm−3 naphthalene acetic acid (NAA) Shoot buds were proliferated from white nodular cultures on hormone free medium Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm−3 2iP + 05 mg dm−3 NAA Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium Among four concentrations of sucrose used, 30 g dm−3 speeded up the bud proliferation more than 10, 20 and 40 g dm−3 However, the largest shoot buds were observed with 40 g dm−3 sucrose Solidification of culture media by 175 g dm−3Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm−3Phytagel