• Journal of Veterinary Clinics
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• v.22 no.4
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• pp.377-381
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• 2005

We tried to extract bone morphogenetic protein (BMP) from the freeze-dried bovine cortical bone (FBCB) for bone graft, which were defatted with chloroform-methanol for 20 days, freeze-dried at $-80^{\circ}C$ for 7 days and sterilized by ethylene oxide gas. Two kg of FBCB were pulverized in a wheel mill to $0.5-2.0mm^3$ cubic in size. The bone particles were demineralized in 0.6N HCI for 10 days at chloroform-methanol$4^{\circ}C$ and defatted with chloroform-methanol for 6 hours at room temperature, which was going to be defatting and demineralized cortical bone (DDM). For extracting BMP, DDM was agitated continuously through 72 hours with magnetic stirrer at $4^{\circ}C$ into 12 times of volume of 6 M guanidine hydrochloride (Gdn-HCl) solution containing proteinase inhibitors to protect BMP such as 2mM N-ethylaleimide, 1mM iodoacetic acid, 1mM phenylmethylsulfonyl fluoride and a sterilizer, 1mM sodium azide. The extraction procedure was repeated for three times. All extracted solution was centrifuged at 10,000 rpm for 30 min and then, the supernatant was dialyzed with 12 times of volume of deionized water at $4^{\circ}C$ for 24-72 hours, which cut off below 6,000-8,000 molecular weight. The dialyzed specimen contained crude-BMP was centrifuged, freeze-dried, and weighted. Through these processing, we could obtained $84.9\%$ as FBCB, $17.8\%$ as DDM and $0.71\%$ as crude-BMP from the wet cortical bone without cancellous bone, marrow and muscles. The crude-BMP were obtained $68.3\%$ from the first extraction, $29.6\%$ from secondary and $2.1\%$ from tertiary, respectively. It was suggested that high yield of crude-BMP migth be explained by three-time repetition of the extraction processing for crude-BMP with Gdn-Hcl sol.

• Journal of Korean Society of Forest Science
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• v.103 no.1
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• pp.72-79
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• 2014

Adventitious buds were produced from the cultures of mature zygotic embryos of Larix kaempferi with the highest frequency in Quoirin & Lepoivre (LP) medium containing 1.0 mg/L zeatin (76.1%). The effective treatments for inducing adventitious shoots growth above 2 mm were shown in Litvay (LM) medium with 0.5 mg/L zeatin (75.2%) or LP medium with 2.0 mg/L zeatin (70.2%), respectively. In experiment with half strength salts medium for induction of the adventitious buds, the effective treatments were obtained from 1/2LP medium with 1.0 (83.3%) or 2.0 mg/L (81.7%) zeatin, respectively. However, the best adventitious shoot growth more than 2 mm appeared in 1/2LM medium with 1.0 mg/L zeatin (66.7%). In experiment with half strength salts medium for induction of the adventitious buds, the effective treatments were obtained from 1/2LP medium with 1.0 (83.3%) or 2.0 mg/L (81.7%) zeatin, respectively. However, the best adventitious shoot growth more than 2 mm appeared in 1/2LM medium with 1.0 mg/L zeatin (66.7%). In experiment of subsequent treatment with various cytokinins for induction of the adventitious buds, the best one (52.9%) was obtained from 1.0 mg/L zeatin for 2weeks, and then subcultured to the medium with 1.0 mg/L thidiazuron (TDZ). The effect of ethylene synergist or inhibitor on adventitious buds induction was examined. The highest rate (34.6%) of adventitious buds marked from the treatments of 1.0 mg/L zeatin+2.0 mg/L MGBG (methylglyoxal bis-[guanylhydrazone]). And the highest no. of adventitious buds(1.5/explant) was shown in the medium with 1.0 mg/L zeatin+2.0 mg/L $CoCl_2$.