MSB Medium Research Articles

In vitro and ex vitro rooting of micropropagated shoots using three green ash ( Fraxinus pennsylvanica ) clones

Shoot multiplication using seedling materials was achieved by subculture on Murashige and Skoog salts with Gamborg's B5 vitamins (MSB5) medium containing a combination of 5 μM 6-benzyladenine (BA), 5 μM thidiazuron (TDZ), and 1 μM indole-3-butyric acid (IBA) with three green ash (Fraxinus pennsylvanica) clones, SD1009 (South Dakota origin), SD2002 (South Dakota origin), and KA2018 (Kansas origin). Shoots were rooted using in vitro and ex vitro methods. For in vitro rooting studies, elongated shoots were transferred to rooting plugs supplied with liquid MSB5 medium containing a 3×3 factorial arrangement of two different auxins, α-naphthaleneacetic acid (NAA) and IBA, at three concentrations (0, 5, and 10 μM). The most effective treatment for in vitro root number, root length, and shoot height was 5 μM IBA. The three clones also were tested for ex vitro rooting using a quick dip in 1 mM NAA, 1 mM IBA, or control (no auxin). The maximal ex vitro rooting response occurred when shoot explants of the three clones were dipped in 1 mM IBA. Significant clonal differences were noted in response to in vitro and ex vitro rooting treatments. Rooted plantlets were acclimated to the greenhouse.

Somatic Embryogenesis and Long Term High Plant Regeneration from Barley (Hordeum Vulgare L.) Using Picloram

Long term high plant regeneration was obtained from embryogenic callus induced from immature embryo explants of Hordeum vulgare cv RD-57 cultured on MSB medium supplemented with Picloram (2 and 4mg I-1). In the subsequent subcultures different types of calli were recognised which could be distinguished from each other on the basis of their morphology, growth and plant regeneration ability. Best growth and maintenance of embryogenic state of callus was achieved on MSB medium supplemented with Picloram (2 mgl-1). For plant regeneration, embryogenic callus was transferred to hormone free MSB medium. Regenerated plantlets were rooted either on hormone free MSB medium or MSB medium supplemented with IAA (0.5 mgl-1). The regenerated plants were transferred to field conditions where they matured and set seeds. Histological studies of embryogenic callus revealed all the stages of typical somatic embryogenesis. In addition, atypical somatic embryos and secondary somatic embryogenesis was also observed and occasionally de novo shoot morphogenesis was seen in the same cultures.

Effects of thidiazuron and benzyladenine on axillary shoot proliferation of three green ash (Fraxinus pennsylvanica Marsh.) clones

Mature seeds of three green ash (Fraxinus pennsylvanica Marsh.) clones, SD1009 (South Dakota origin), SD2002 (South Dakota origin), and KA2018 (Kansas origin) were cut to remove the apical portion and germinated on Murashige and Skoog (1962) salts with B5 vitamins (Gamborg et al., 1968) (MSB5) medium without plant growth regulators. Stable axillary shoot establishment was achieved for all three clones by subculture on MSB5 medium containing a combination of 5 μM thidiazuron (TDZ), 5 μM 6-benzyladenine (BA), and 1 μM indole-3-butyric acid (IBA). Following shoot establishment, axillary shoots were placed on MSB5 medium containing a single treatment of TDZ (1, 5, 10, 20, or 40 μM) or BA (1, 5, 10, 20, 40, or 80 μM). Concentration of TDZ and BA significantly affected shoot biomass (total dry weight of axillary shoots), with 10 μm TDZ or 40 μm BA providing maximum shoot proliferation with all three clones. Significant clonal differences also were noted in the proliferation of axillary shoots, with clone SD1009 exhibiting the highest axillary shoot proliferation. Axillary shoots were rooted under ex vitro conditions and acclimatized to the greenhouse.

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10(5)/ml. The protoplasts began to divide in 3-5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3-5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1-0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.

Plant regeneration from seedling-derived callus of dodder ( Cuscuta trifolii Bab. et Giggs)

Callus was induced and plants were regenerated for Cuscuta trifolii, the obligatory parasite of Medicago sativa. Callus was initiated on young seedlings grown in liquid culture medium consisting of Murashige and Skoog basal salts, Gamborg's vitamins (MSB5), 5 mg/l kinetin and 0.75 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The structure of callus tissue depended on the carbohydrates supplied. Friable, fast growing calluses were obtained in the presence of maltose and sorbitol combined (1.5-1.5%) and was maintained on corresponding agar-solidified medium for over a year. Plants were regenerated from calluses grown on solidified MSB5 medium that either lacked growth regulators or contained 2 mg/l kinetin. Embryo-like forms developed continuously from this culture for over 5 months. The detached embryo-like structures were placed in liquid MSB5 medium containing 2 mg/l kinetin and they grew as seedlings would under the same conditions. Both embryo-like structures and seedlings developed aggregate structures from which shoots grew. In this way, Cuscuta plants could be maintained in vitro without the presence of a host.

Plant Recovery from Embryonic Axes of Deteriorated Peanut Seed for Germplasm Renewal1

Abstract Embryo axes explants from deteriorated seed of peanut (Arachis hypogaea L.) were incubated at a 16 hr photoperiod at 26 C on an MSB5 medium containing MS salts, B5 vitamins, 20 g/L sucrose, and 8 g/L agar. Five to 8-wk-old plants regenerated from embryonic axes were transplanted to Jiffy pots in the greenhouse. Thirty-two samples of deteriorated seed between 2 and 32 yr old were evaluated. Significant differences in organogenesis were observed between different seed accessions. Shoots and roots were recovered from 74 and 36%, respectively, of embryonic axes explants. In the same experiment, seed producing plants were recovered from 14- to 31-yr-old deteriorated seed of E-2, PI 275704, Macrocarpa, G33, G34, G64, Strain No. 5, TMV 3, PI 290608, PI 295981, Sekelembwe, PI 298879, PI 337300, and PI 371850, by in vitro rescue of embryonic axes, while no plants were recovered from seed of 31 seed accessions germinated in the field. The in vitro rescue of embryonic axes can significantly increase the recovery of germplasm from deteriorated seed of peanut.

Transgenic plants of rutabaga (Brassica napobrassica) tolerant to pest insects

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B5 Vitamins) containing 6-BA 3mg/1 for 2-3 days, and transferred onto selective medium (MSB with kanamycin 50-100 mg/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants were obtained. About 60% of the plants showed high NPT II activity. Southern blot hybridization showed that some of the plants gave a positive signal with the insecticidal crystal protein gene (cry IA gene) probe, and exhibited tolerant to insects such asPieris rapae (cabbage caterpillar) in leaf feeding experiments. Kanamycin-resistance and insect-resistance were maintained in the progeny.

Plants regenerated from mesophyll protoplasts of white mulberry

Morus alba(white mulberry) mesophyll protoplasts were isolated from leaves of 30-45 day old sterile shoots, with protoplast yields of 2.5 × 107 g−1/F.W. after purification. The protoplasts were cultured in a modified K8P liquid medium containing 0.2 mg/L 2,4-D(2,4- Dichlorophenoxy acetic acid), 1 mg/L NAA(Naphthyl acetic acid) and 0.5 mg/L BA(6-benzylaminopurine). A low plating density (5 × 104/ml) proved to be favourable to the division of protoplast-derived cells. The first division occurred 4 days after culture, and the division frequency reached 24% at 10 days. A number of cell colonies and microcalli formed in 6 weeks. The microcalli were transferred onto MSB medium with 0.5 mg/L NAA and 0.5 mg/L BA for further proliferation. Shoot forrgation was initiated when the calli of 3-4 mm in size were transferred onto MSB differentiation medium with 0.1 mg/L NAA and 1 mg/L BA. The frequency of shoot formation was 35%. The shoots of 4-5 cm in height were excised from the callus and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA. After transplantation into pots, the regenerated plants grew vigorously in the phytotron.

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 µM]), picloram (1.2 mg/liter [5.0 µM]), and kinetin (2.1 mg/liter [10 µM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 µM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 µM]: 0.5 mg/liter [2.22 µM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 µM) and 20 mg/liter (148.04 µM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 µM]) stimulated bulb formation by 30 days in culture.

Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response

Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1-2 mm diameter colonies were formed in 5-6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l(-1) each of 2,4-D, BA and KN and 500 mg l(-1) CH for further growth. Once the colonies had become green, compact and nodular, and were 8-10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8-12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45-50% were obtained in liquid medium and 55-60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1-2 mm in size) were obtained in 5-6 weeks, which upon transfer to MSB medium with 0.5 mg 1(-1) each of 2,4-D, BA, Kn and 500 mg 1(-1) CH produced compact green calli in 4-6 weeks. After 3-4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1(-1) each of BA, Kn, ZT, 0.1 mg 1(-1) NAA and 500 mg 1(-1) CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA3 enhanced shoot regeneration up to 30%. Shoots of 0.5-1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1(-1) TH and 0.5 mg 1(-1) GA3 for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2-3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1(-1) IBA or 0.5 mg 1(-1) NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.

Somatic Embryogenesis and Plant Regeneration from Leaves of Dendranthema grandiflora

Somatic embryogenesis from leaf midrib explants of Dendranthema grandiflora Tzvelev. `Iridon' cultured on modified Murashige and Skoog basal medium (MSB) containing 1.0 mg 2,4-D and 0.2 mg BA/liter was influenced by light and sucrose concentration. Somatic embryos formed directly from explants when cultured on medium containing 9% to 18% sucrose and incubated first in the dark for 28 days, followed by 10 days in light, and then returned to the dark for 14 days. Embryogenesis did not occur in continuous darkness and was drastically reduced when explants were incubated in light only. The most embryos were formed on medium containing either 12% or 15% sucrose; lower concentrations stimulated shoot and root development. Light also mediated embryogenesis from leaf explants of 'other cultivars. White-opaque or occasionally light-green cotyledon-stage somatic embryos germinated on MSB medium without growth regulators but containing 3% sucrose. Twelve of the 23 cultivars evaluated produced somatic embryos, but plants were recovered from only five. Regenerated plants were phenotypically similar to parent plants in growth habit, leaf morphology, and flower color. Chemical names used: N- (phenylmethyl)-1 H- purine-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).

Bacteriological Examination of Fissure Plaques from Seriously Mentally Retarded Adults

The microfloras of fissure plaque samples removed from a total of 23 seriously mentally retarded adults, resident in two institutions, were examined and compared with the fissure microfloras found in 17 control subjects who were employees in both institutions. The samples were collected from both an intact fissure and a fissure with brown-stained defective enamel surface of the premolars of the lower jaw. Mutans streptococci, selected with MSB medium, were recovered from only one of the defective fissures of 13 patients who became retarded younger than 2 years of age, whereas 14 defective fissures of 17 controls harbored mutans streptococci. Four of 8 patients who became retarded older than 3 years of age harbored mutans streptococci. Lactobacilli were infrequently recovered from both patients and controls. The results are discussed in terms of the microbiological aspect of caries development in adults who became mentally retarded when young.

Plant regeneration from protoplasts of immature cotyledons of Glycine soja Sieb. et Zucc.

Glycine soja Sieb. et Zucc. (a wild species of soybean) protoplasts were isolated from immature cotyledons and initially cultured in a modified K8P medium supplemented with 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/l naphthalene acetic acid (NAA) and 0.4 mg/l benzyl adenine (BA). The protoplasts started to divide after 3 days of culture. The division frequency of protoplast-derived cells counted at 12 days reached 36.8%. After 6 weeks, cell colonies formed and transfer to gelrite-solidified K8 cell medium with 0.1–0.2 mg/l NAA and 0.5–2 mg/l BA supported further proliferation and callus growth. When calli of 2–3 mm in size were subcultured on MSB growth medium with 0.2 mg/l NAA and 0.5 mg/l BA, approximately 15% of them developed into compact and nodular calli. Shoot buds were regenerated on the surface of the nodular calli with a frequency of 25%, when the calli were placed on MSB medium with 0.1 mg/l indole-3-acetic acid (IAA) and 0.2–0.5 mg/l BA. Whole plants were regenerated upon transfer of 3–4 cm shoots to 1 2 Murashige and Skoog (MS) medium with 0.2 mg/l indole butyric acid (IBA). Twenty-four complete plants have been obtained and seven of them transplanted into pots are growing well in the phytotron.

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3-5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2-3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.

Chemical Factors Controlling Morphogenesis of Petunia Cells Cultured in vitro

Calluses of different types were obtained from in vitro cultured stem segments of Petunia inflata R. E. Fries and Petunia hybrida L. (var. Cascade and Rose du Ciel) using different agarified media. A cell suspension from each type of callus was prepared in a liquid medium, the composition of which was the same as for the primary cultures (MI: MSa medium + 2,4-D 1 mg/l; MII: MSb medium + 2,4-D 1 mg/l; MIII: MSa medium + IAA. 1 mg/l + BA 200 μ g/l + GA 3 200 μ g/l). Each suspension culture was then transferred and grown in MSa medium + 2,4-D 100 μ g/l + kinetin 100 μ g/l (MIV). The morphogenetic potential of suspended cells depended upon the medium from which it had originally been derived. Cells derived from Ml gave somatic embryos, from MII gave roots whilst those from MIII gave undifferentiated calluses and shoot primordia. Most of the somatic embryos were morphologically similar to zygotic embryos and gave rise to new plants.

Isolation and screening of rhizobacteria from soil in Ngawi, East Java,as candidates of agent for liquid organic fertilizer production

Abstract. Imamuddin H, Dewi TK, Antonius S. 2015. Isolation and screening of rhizobacteria from soil in Ngawi, East Java as candidates of agent for liquid organic fertilizer production. Nusantara Bioscience 7: 107-111. A series of activities have been done to find indigenous microbes from Ngawi, East Java, as candidates for living organic fertilizers. The study was started with sampling of soils from 17 locations in Ngawi. Then, the samples were isolated to get population of soil microbes, including total, phosphate-solubilizing, N-fixing, and IAA-(Indol Acetic Acid)-producing bacteria. Five soil samples were used to test bacterial growth in several concentration of propoxur. Method of isolation used in this study was enrichment culture and the growth was determined using spectrophotometer at 436 nm wave length. The resuts showed that the populations of total and IAA-producing bacteria in Ngawi soil were relatively high, namely 106 CFU/g soil, but the population N-fixing bacteria was only 10-5, and no phosphate-solubilizing bacteria could grow. Twenty one isolates of bacteria were found, 16 of which postively produced IAA and 5 isolates of which could grow in propoxur medium. The highest production of IAA was found in sample number 6.3 with a concentration of 123.535 ppm and isolate H-2-NG (sample number 2) could grow at concentration of 1000 ppm (MM medium)-3000 ppm (MSB medium). It is hoped that these isolates can be used as candidates of agent for liquid living organic fertilizers.

<i>In</i> vitro Plant Regeneration of Four Local Varieties of Chickpea (<i>Cicer arietinum</i> L.) Grown in Bangladesh

In vitro regeneration system was developed through direct organogenesis from decapitated mature embryo explants of locally grown four chickpea varieties, namely, Barichhola-4, Hyprochhola, Binachhola-3 and Binachhola-4. Best response towards multiple shoot regeneration was obtained on MS medium supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn, 0.2 mg/l NAA along with double concentrations of CaCl2 and NH4NO3. However good shoot health and expanded leaf was found on MS medium containing 1.0 mg/l kn. Apart from this, few experiments were conducted with decapitated embryo attached cotyledon. Using this explants highest number of multiple shoots were obtained on MSB medium containing 4× micronutrients of MS medium with 3.0 mg/l BAP and 0.04 mg/l NAA in all four varieties. Shoots regenerated on 1.0 mg/l kn supplemented medium showed good response towards rooting on MS medium supplemented with 0.2 mg/l IBA in all four varieties. It was observed that micrografting is an alternative technique to in vitro rooting in chickpea. Key words: In vitro regeneration; Decapitated embryo; Chickpea. DOI: http://dx.doi.org/10.3329/bjsir.v46i3.9047 BJSIR 2011; 46(3): 379-384