Liquid Murashige And Skoog Medium Research Articles

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.).

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 106 cells/g fresh weight cells and then cultured at a concentration of 2.5×105 cells/ml in NH4NO3-free Murashige and Skoog (MS) medium supplemented with 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 µM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 µM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 µM kinetin or 1 µM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants.

Varietal Differences in Plant Regeneration from Solid and Suspension Cultures in Onion (Allium cepa L.).

Seeds of 22 onion (Allium cepa L.) cultivars were cultured on solid Murashige and Skoog (MS) medium containing 50 μM 4-fluorophenoxyacetic acid (4-FPA) and 1 μM N6 (2-isopentenyl) adenine (2iP). Callus formation was observed in all cultivars. Some of the calli in solid culture were transferred into liquid MS medium containing 50 μM 4-FPA and 1 μM 2iP and were cultured in suspension. After subculturing in a liquid medium of 0.2M of sucrose, light yellow and large (LL) type cell clumps were obtained. To investigate the varietal differences in plant regeneration from solid and suspension cultures, the calli in solid culture and LL type clumps in suspension culture were transferred onto solid MS medium containing 1 μM 2iP. Plantlets were regenerated directly from the calli and the LL type clumps. The frequency of plantlet regeneration from suspension cultures tended to be higher than that from solid cultures. Significant differences were observed among the cultivars in the frequencies of plantlet regeneration from solid cultures and from suspension cultures. After suspension culture, 'Kurenai' showed the highest regeneration frequency (98%).

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai.

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 µM kinetin or zeatin, but was highest on medium containing 4.5 µM 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 µM 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 µM 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse.

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.).

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1-10 μM and α-naphthaleneacetic acid (0-0.5 μM showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 μM benzyl adenine and 0.25 μM α-naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 μM indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.

In vitro multiplication ofVanilla planifolia using axillary bud explants.

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N6-benzyladenine (BA, 2 mg l-1) and α-naphthaleneacetic acid (NAA, 1 mg l-1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l-1 and NAA at 0.5 mg l-1 for 2-3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.

Production of Somatic Hybrid between Cultivars of Sweet Potato, Ipomoea batatas(L.) Lam. in the Same Cross-incompatible Group.

This is the first report on successful production of somatic hybrid between cross-incompatible cultivars of sweet potato, lpomoea batatas (L.) Lam. The protoplasts of sweet potato cultivars, Koganesengan and Bitambi, belonging to the same cross-incompatible B group and not crossing with each other, were fused with Polyethylene Glycol (PEG) solution and then cultured in modified liquid MS (Murashige and Skoog 1962) medium containing 0.05 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg/l kinetin. Forty-five plants were obtained from the calli on the hormone-free medium by using the callus proliferation medium either supplemented with 2, 4-D and kinetin or abscisic acid (ABA), or supplemented with naphthalene aceic acid (NAA) and 6-benzylaminopurine (BAP). Two plants were considered to be the somatic hybrids by analyzing their morphology, chromosome numbers and random amplified polymorphic DNA (RAPD) markers. The hybrids showed intermediate characters in leaf shape and leaf color between the two parents and had chromosome numbers that correspond to the sum of their parents (2n= 12x (2n+2n)= 180). By RAPD assay, they expressed some of the bands from both parents. They grew slowly in the field. One of them had the pollen fertility of 12.2 % when grafted on the morning-glory for flower induction. No seed was obtained by crossing with pollen of Kyushu No. 30 (cross-incompatible C group cultivar).

Normalization of asparagus somatic embryogenesis using a maltose-containing medium

Summary Embryogenic calli derived from internal buds were cultured and maintained on Murashige and Skoog (MS) medium containing 2 mg/L 2,4-D, 3 % sucrose and 0.2 % gellan gum. Normal somatic embryos were induced from the embryogenic calli by pretreating with ½MS liquid medium for 7 days, sieving through 600 μm stainless steel mesh and then partially desiccating on plant growth regulator-free MS medium containing a high concentration of gellan gum (1.0% w/v) for 1 week. Furthermore, use of maltose as a sugar and/or osmoticum in the embryo induction medium promoted development of normal somatic embryos. Using these methods, non-vitrified bipolor embryos were induced 30 days after transfer (approximately 1,500 embryos per 0.1 mL packed cell volume). The somatic embryos induced on maltose-containing medium exhibited a much higher germination rate (more than 80 %) with cold treatment (14 days at 4 °C) than that induced on sucrose-containing medium.

Tannins and Related Compounds in Callus and Root Cultures of Cornus capitata.

Callus and adventitious root cultures of Cornus capitata were established and the production of tannins and related compounds was determined in various culture conditions (media, plant growth regulators, light, etc.). Calli produced mainly procyanidins such as (+)-catechin and procyanidin B-3 on Murashige and Skoog (MS) and Woody Plant (WP) media. The maximum content of (+)-catechin (0.1%, as dry weight) was observed in the calli cultured on WP medium supplemented with 0.1mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1mg/l benzyladenine (BA), or 2.0mg/l naphthaleneacetic acid (NAA) and 0.1mg/l BA in the light. In contrast, adventitious roots cultured in MS liquid medium supplemented with 3.0mg/l indoleacetic acid (IAA) produced a high level of gallotannins such as 1, 2, 3, 6-tetra-O-galloyl-β-D-glucose and 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose.

In Vitro Shoot Development of Eucalyptus citriodora on Rockwool in the Film Culture Vessel under CO2 Enrichment

An efficient system for growingin vitro plantlets ofEucalyptus citriodora Hook was developed. In the conventional closed system of culture with 2% sugar-containing gellan gum Murashige and Skoog (MS) medium supplemented with 0.02 mg/l indole-3-isobutyric acid, a serious defoliation of shoots was observed after three weeks. In contrast, plantlets grown on the sugar-free MS medium in the aerated bottle under 3,000 ppm CO2 enriched condition did not show any defoliation. A marked enhanced growth of plantlets and no defoliation were observed on rockwool with the sugar-free liquid MS medium in the “Culture Pack”, made of fluorocarbon polymer film, under CO2 enriched condition. CO2 enrichment for this sugar-free “Culture Pack”-Rockwool system was also found to contribute to an improved growth of the plants in acclimatization.

Micropropagation ofDalbergia sissoo from nodal explants of mature trees

A method for micropropagation ofDalbergia sissoo has been developed. Single node segments obtained from coppice shoots of a mature tree (20 - 25 year old) produced 3-4 shoots per explant on Murashige and Skoog (MS) medium containing 4.4 x 10-6 M benzylaminopurine (BAP) and 4.4 × 10-7 M of Β-naphthoxy acetic acid (NOA) (shoot multiplication medium) within 4 weeks. Thein vitro regenerated shoots were 3 - 4 cm in length and provided 2 to 3 culturable nodal segments which on shoot multiplication medium again produced 3-4 shoots. Following this procedure 18-24 shoots were produced from single nodal segment within 60 d. 80 % of the shoots directly produced five roots when they were firstly treated with MS medium supplemented with 10-5 M indole-3-butyric acid (IBA) and subsequently transferred to half strength liquid MS medium containing 1 % activated charcoal followed by half strength liquid MS free hormones, vitamins and activated charcoal. Thein vitro raised plants were hardened for survival after transplantation to soil by exposing them to various humidity conditions, gradually from higher to low, with nearly 100 % transplant success.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Efficient In vitro Mass-propagation of Shiode(Smilax oldhami Miq.) through Liquid Culture.

Shiode is a wild and edible plant whose flavor is similar to that of asparagus (Asparagus officinalis L.). The present experiments were implemented to improve the in vitro propagation system of Shiode. Internodal explants were cultured to derive protocorm-like bodies (PLBs) on Murashige and Skoog (MS) medium at pH 5.7. The MS medium was supplemented with 30g/l sucrose, 0.1mg/l α-naphthaleneacetic acid (NAA) and 0.1mg/l 6-benzylaminopurine (BAP), and solidified with 8g/l agar. The PLBs that were induced on the solid MS medium propagated very rapidly when they were transferred into the 1/2 MS liquid medium at pH 5.7 supplemented with 20g/l sucrose, 1.0mg/l NAA and 0.1 or 1.0mg/l BAP. The liquid MS medium was renewed periodically. The single PLBs were separated from the mother PLB-clusters by a surgical knife, and were used to regenerate the descendant PLB-clusters. The secondary PLBs grew adventitious shoots that formed roots on the solid hormone-free MS medium after subculture for 2 weeks on the solid MS medium supplemented with 0.5, 1.0 and 2.0mg/l NAA. The plantlets were acclimatized following standard procedures.

SOMATIC EMBRYOGENESIS IN CELL SUSPENSION CULTURE OF COWPEA (VIGNA UNGUICULATA (L.) WALP)

Embryogenic callus was induced from seedling leaf explants of cowpea (Vigna unguiculata (L.) Walp. cv. C152 on Murashige and Skoog (MS) medium containing 2.0 mg 1−1 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was noticed when this callus was transferred to MS liquid medium supplemented with 2 mg 1−1 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical or filamentous proembryos. Subsequent divisions in the proembryo led to globular, heart, torpedo-shaped, and cotyledonary-stage somatic embryos. Tiny plantlets were obtained by transferring the cotyledonary-stage somatic embryos to MS liquid medium containing 0.5 mg 1−1 2,4-D.

Improved culture conditions for somatic embryogenesis using an aseptic ventilative filter in eggplant ( Solanum melongena L.)

Embryogenic calli were induced from a cotyledon cutting of Solanum melongena L. on Murashige and Skoog (MS) solid medium supplemented with 50 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Calli were proliferated in MS liquid medium supplemented with 50 μM 2,4-D. Non-vitrified somatic embryos were formed and efficiently developed into cotyledonary embryos on transfer to MS hormone-free medium with high concentration (1%) of gelrite in a culture vessel capped with an aseptic ventilative filter. Somatic embryos developed into normal plants when transferred to half-strength MS solid medium without hormones. In contrast, vitrified and swollen embryos were formed on the same medium with 0.2% gelrite in culture vessels capped with aluminum foilm could not germinate normally and developed in part into callus tissue. The established system here was applied to 10 cultivars, and eight of them could form non-vitrified embryos that developed into normal plants. The effects of an aseptic ventilative filter on somatic embryogenesis are discussed.

Somatic embryogenesis of tetraploid plants from internodal segments of a diploid cultivar of Asparagus officinalis L. grown in liquid culture

Calli were induced from internodal segments of seedlings or young spears of Asparagus officinalis L. on Murashige and Skoog (MS) liquid medium with 3 mg/l napthaleneacetic acid (NAA), 1 mg/l kinetin (KN) and 3% sucrose. The cultures were grown on a rotary shaker at 100 rev./min at 25°C in a light/dark cycle of 16/8 h (6000 lux from fluorescent lamps). Minicalli in the liquid medium were harvested by sieving through a stainless steel mesh of 1 mm pore size and washed twice with 50 ml of plant growth regulator-free MS liquid medium. An aliquot of 0.6 ml packed cell volume calli were added to 30 ml of PGR-free MS liquid medium and cultured under the same conditions as for callus induction. Somatic embryos obtained 1 month after transfer to PGR-free medium germinated normally with shoots and roots after transfer to Gellan Gum-solidified MS medium. All of the regenerated plants were tetraploid. They were successfully grown in pots containing soil in a greenhouse after acclimatization in an incubator.

TRANSIENT GENE EXPRESSION IN SPINACH CALLUS TRANSFORMED WITH AGROBACTERIUM TUMEFACIENS

The objective of this study was to determine the efficacy of Agrobacterium tumefaciens in transforming spinach (Spinacia oleracea L.) callus. Callus was induced from leaf disks of `Baker' on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 kinetin and 0.5 mg L-1 2,4-D. Callus was cut into 2-mm pieces, and 0.5 g of callus was placed in each 250-ml flask which contained 20 ml of MS liquid medium. The suspension cultures were inoculated with 100 μl of an overnight culture of A. tumefaciens harboring pMON 9749 (provided by S. Rogers, Monsanto Co., St. Louis), a plasmid cointegrated with kanamycin resistance and β -glucuronidase (GUS) genes. After coculturing for 2 days at 22C with shaking at 100 rpm, the medium was replaced with selection medium containing (in μg/ml) 75 kanamycin, 100 cefotaxime, and 200 carbenicillin and maintained for 3 weeks. Transient expression of GUS gene in transformed cells was detected with X-glu assay. This method resulted in a high level of transformation and provides the first report of transformation in spinach. This study was funded by a grant (92-B-32) from the Arkansas Science & Technology Authority.

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.

An efficient method for selecting embryogenic callus from Lycium chinensis L. cell culture

Cell culture of Lycium chinensis L., Solanaceae, was used to select a cell line with high regeneration potential through somatic embryogenesis. Friable callus was induced from shoot tips and grown in Murashige and Skoog (MS) medium containing 0.5 mg/l naphthalene acetic acid (NAA) and 0.2 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) (MS3 medium) until transfer to MS liquid medium without plant hormones (MSO medium). Pale green granules of compact callus were selected from this culture as the HE cell line. The selected HE cell line, which was sub-cultured on the MS3 agar medium for at least three passages, was shown to be more embryogenic than the original (LE) cell line. Histological studies indicated somatic embryogenesis.

Calli and suspension cultures for biomass production ofEuphorbia characias L. subsp.characias

Friable calli and cell suspension cultures were obtained from leaf segments ofEuphorbia characias L. subsp.characias, in Murashige and Skoog (MS) basal medium supplemented with 1g.l−1 casein hydrolyzate (CH), 5mg.l−1 ascorbic acid, 1.0mg.l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.75mg.l−1 benzyl adenine (BA). The highest callus specific growth rate (μ=0.085.day−1), calculated for 1 year old calli cultures, was obtained with 0.25 mg.l−1 2,4-D and 0.50mg.l−1 BA. Suspension cultures started with an inoculum of 8.0×104 cells.ml−1 in supplemented liquid MS medium, gave a specific growth rate μ=0.256.day−1.

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.