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An efficient protocol for peanut (Arachis hypogaea L.) transformation mediated by Agrobacterium rhiz

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Romanian Biotechnological Letters Vol. 14, No. 5, 2009, pp. 4641-4647 Copyright © 2009 University of Bucharest Printed in Romania. All rights reserved Romanian Society of Biological SciencesORIGINAL PAPER An efficient protocol for peanut (Arachis hypogaea L.) transformation mediated by Agrobacterium rhizogenesReceived for publication, March 3, 2009Accepted, October 20, 2009JONG SE KIM1, SOOK YOUNG LEE2, SANG UN PARK3,*1Department of Biology, 2Research Center for Oral Disease Regulation of the Aged, ChosunUniversity, 375 Seosuk-Dong, Dong-Gu, Gwangju, 501-759, Korea3Division of Plant Science and Resources, Chungnam National University, 220 Gung-Dong,Yuseong-Gu, Daejeon, 305-754, Korea* Corresponding Author. Phone: +82-42-822-2631. Fax: +82-42-822-2631.E-mail: supark@cnu.ac.kr.AbstractThe Agrobacterium-mediated transformation of hairy roots creates a rapid and simple means to introduce and express foreign genes in plant cells that are capable of synthesizing specific natural products. We describe a method for the production of transgenic hairy root cultures of peanut (Arachis hypogaea L.) using Agrobacterium rhizogenes R1000, a strain with the binary vector pBI121. We evaluated the stable transformation of kanamycin-resistant hairy roots by determining the integration of the NPTП gene into the plant genome, the histochemical localization of GUS activity, the presence of GUS mRNA, and the level of GUS enzyme activity. Transgenic root culture ofA. hypogaea is a simple, reliable and well-defined model system to investigate the molecular andmetabolic regulation of resveratrol biosynthesis, and to evaluate the metabolic engineering potential of peanut.Keywords:Arachis hypogaea L., Agrobacterium rhizogenes, hairy root, peanut, transformationIntroductionPeanut (Arachis hypogaea L.) is an annual oil seed legume that is native to South America, but is currently grown in diverse environments world-wide [22]. Peanuts, grapes, and Japanese knotweed are important sources of resveratrol (trans-3,5,4'-trihydroxystilbene) [4; 24]. Resveratrol has anti-inflammatory, antioxidant, and anti-infective properties and also has potential therapeutic efficacy against breast cancer, prostate cancer, and neuroblastoma [1; 15; 12; 2].In many plants, hairy root cultures have proven to be an efficient production system for secondary metabolites. Such cultures have genetic and biochemical stability, rapid growth rate, and the ability to synthesize natural compounds at levels comparable to those of intact plants [9; 23; 7; 8].Investigation of the molecular mechanisms that regulate resveratrol biosynthetic enzymes in peanut requires establishment of protocols for its efficient and stable genetic transformation. Agrobacterium rhizogenes-mediated transformation of hairy roots provides a rapid and simple means to introduce and express foreign genes in plant cells that are capable of synthesizing specific secondary metabolites, such as resveratrol. Recently we reported five different strains of Agrobacterium rhizogenes differing in their ability to induce peanut hairyJONG SE KIM, SOOK YOUNG LEE, SANG UN PARKroots and also showed varying effects on the growth and resveratrol production in hairy root cultures [11]. In this paper, we describe a protocol for introducing foreign genes into peanut hairy root cultures using A. rhizogenes.Materials and MethodsPlant materialSeeds of A. hypogaea were surface-sterilized with 70 % (v/v) ethanol for 1 min and 2 % (v/v) sodium hypochlorite solution for 10 min, then rinsed three times in sterilized water. Four seeds were placed on 25 ml of agar-solidified culture medium in Petri dishes (100 x 15 mm). The basal medium consisted of salts and vitamins of MS (Murashige and Skoog) [18] medium and solidified with 0.7 % (w/v) agar. The medium was adjusted to pH 5.8 before adding agar, and then sterilized by autoclaving at 121 °C for 20 min. The seeds were germinated in a growth chamber at 25 °C under standard cool white fluorescent tubes with a flux rate of 35 µmol s-1 m-2 and a 16-h photoperiod.Preparation of Agrobacterium rhizogenesThis experiment was conducted using the binary vector pBI121. The pBI121 plasmid has a CaMV 35S promoter-GUS gene fusion and the neomycin phosphotransferase (NPT II) gene as a selectable marker. This binary plasmid was transferred into Agrobacterium rhizogenes R1000by electroporation. The culture of A. rhizogenes was initiated from glycerol stock and grown overnight at 28 o C with shaking (180 rpm) in liquid Luria-Bertani medium containing 50mg/L kanamycin, to mid-log phase (OD600 = 0.5). The A. rhizogenes cells were collected by centrifugation for 10min at 250g and resuspended in liquid inoculation medium (MS salts and vitamins containing 30 g/L sucrose). The A. rhizogenes cell density was adjusted to given an A600 of 1.0 for inoculation.Establishment of transgenic hairy root culturesExcised leaves of A. hypogaea from 20-day-old seedlings were used as an explant material for co-cultivation with A. rhizogenes R1000.The excised leaves were dipped into the A. rhizogenes culture in liquid inoculation medium for 15 min, blotted dry on sterile filter paper, and incubated in the dark at 25°C on Phytagar-solidified MS medium. After two days of co-cultivation, the explant tissues were transferred to a hormone-free MS medium containing salts and vitamins, 30 g/L sucrose, 500 mg/L carbenicillin, 50 mg/L kanamycin and 8 g/L agar. Putative transgenic hairy roots were observed emerging from the wound sites within two weeks.Isolated putative transgenic roots (100 mg) were transferred to 30 ml of MS liquid medium, containing 30 g/L sucrose, in 100 ml flasks. Root cultures were maintained at 25°C on a gyratory shaker (100 rpm) in a growth chamber under standard cool white fluorescent tubes with a flux rate of 35 µmol s-1 m-2 and a 16-h photoperiod. After 3 weeks of culture, hairy roots were harvested and the dry weight and resveratrol content were determined. Each experiment was carried out with 3 flasks per culture condition and repeated twice.PCR analysis for NTPIIPlant genomic DNA for polymerase chain reaction (PCR) analysis was extracted as described by Edwards et al. [5]. The tissue (50 mg fresh weight) was homogenized in 200 µl of extraction buffer (0.5% SDS, 250 mM NaCl, 100 mM Tris-HCl, pH 8, and 25 mM EDTA pH 8.0) and centrifuged at 13,200 rpm for 5 min. The supernatant was transferred to a new tube andAn efficient protocol for peanut (Arachis hypogaea L.) transformation mediated by Agrobacterium rhizogenesan equal volume of isopropanol was added. The sample was incubated on ice for 5min and then centrifuged for 10 min at 13,200 rpm. The pellet was dried at 60 °C for 5-10 min and then resuspended in 100 µl of TE (10 mM Tris-HCl, pH 7.4 and 1 mM EDTA, pH 8.0).The sequences of the two primers used to amplify a fragment of the NPTII gene were 5'-TATGTTATGTATGTGCAGATGATT-3' and 5'-GTCGACTCACCCGAAGAACTCGTC-3'. The amplification cycle consisted of denaturation at 95o C for 1min, primer annealing at 55o C for 1min, and primer extension at 72o C for 1min. After 30 repeats of the thermal cycle and final extension 72o C for 5min, amplification products were analyzed on 1% agarose gels. Gels were stained with ethidium bromide and visualized with UV light.Assay of GUS activityPutative transgenic peanut roots were collected and grounded with extraction buffer consisting of 50 mM KPO4 buffer, pH 7.0, 1 mM EDTA, and 10mM-mercaptoethanol. The GUS flurometric assay buffer consisted of 50 mM NaPO4buffer, pH 7.0, 10 mM-mercaptoethanol, 10 mM EDTA, 0.1% (w/v) sodium lauryl sarcosine, and 0.1% (w/v) Triton X-100. 4-Methylumbelliferyl-D-glucuronide was added at a final concentration of 0.44 mg/ml. Assays were performed on 50 L of transgenic shoot tissue extract for 3 h at 37o C and stopped with a 10X volume of 0.2 M Na2CO3. A fluorescence spectrophotometer (model F-2000, Hitachi, Tokyo, Japan) was used to quantify the amount of 4-Methylumbelliferone cleaved from 4-Methylumbelliferyl-D-glucuronide. The protein concentration was determined by the method of Bradford [3] using BSA as a standard.Northern blot hybridizationTotal RNA for gel-blot analysis was isolated from putative transgenic root tissue according to the method of Logemann et al. [16], and 15 µg was fractionated on 1.0% formaldehyde agarose gels before transfer to nylon membrane [20]. RNA gel blot was hybridized with random-primer 32P-labeled [6] full-length GUS-intron. Hybridization was performed at 65o C in 0.25 mM sodium phosphate buffer, pH 8.0, 7% (w/v) SDS, 1% (w/v) BSA, and 1 mM EDTA. Blot was washed at 65o C, twice with 2X SSC and 0.1% (w/v) SDS and twice with 0.2X SSC and 0.1% (w/v) SDS; 1X SSC (= 0.15M NaCl, 0.015M sodium citrate, pH 7.0), and autoradiographed with an intensifying screen at -80o C for 24 h.GUS histochemical stainingHistochemical staining for GUS activity was performed by standard protocol [10] for fixation and the modified method recommended by Kosugi et al. [13] for staining. Hairy roots were fixed in a 0.35% (w/v) formaldehyde solution containing 10 mM MES, pH 7.5, and 300 mM mannitol for 1 h at 20o C, rinsed three times in 50mM sodium phosphate, pH 7.5, and subsequently incubated in 50mM sodium phosphate, pH 7.5, 10 mM EDTA, 300 mM mannitol, pH 7.0, and 1 mM 5-bromo-4-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt for 6 to 12 h at 37o C. Stained tissues were rinsed extensively in 70% ethanol to remove residual phenolic compounds.ResultsWe used A. rhizogenes to develop an efficient protocol for creation of transgenic peanut root cultures. To establish selection conditions, we examined the effect of different concentrations of kanamycin (an aminoglycoside antibiotic that is inactivated by the NPTII gene product) on the induction of peanut hairy roots transformed with wild type A. rhizogenesJONG SE KIM, SOOK YOUNG LEE, SANG UN PARK1000 contained no transformation vectors. Kanamycin at concentrations of 50, 70, and 100 mg/L completely inhibited the induction of hairy roots from explant tissues (data not shown). Therefore, we used 50 mg/L kanamycin for all subsequent procedures.Leaf explants from in vitro-grown peanut plants were infected with A. rhizogenes R1000 that contained the binary vector pBI 121. Two days after infection, we transferred explants to agar-solidified hormone-free selection medium. Wounded explants were highly susceptible to infection by A.rhizogenes R1000. Hairy root initials emerged from wound sites on peanut leaves 2 to 3 weeks after inoculation (Figure 1-A). After 4 weeks, putative transgenic hairy roots of peanut began to grow more rapidly (Figure 1-B). A.rhizogenes R1000 infected about 70% of the explants and induced an average of four to five hairy root initials per explant within 6 weeks.Figure 1. Development of hairy roots from leaf of Arachis hypogaea after inoculation with Agrobacterium rhizogenes strain R1000; Two weeks (A) and four weeks (B) after inoculation. Rapidly growing hairy root culture in MS liquid culture medium (C).Histochemical staining of peanut wild type root (D)An efficient protocol for peanut (Arachis hypogaea L.) transformation mediated by Agrobacterium rhizogenes and hairy root tissue transformed with the GUS gene (E). Kanamycin-resistant roots were induced on the surface of explant tissue within 4 weeks after inoculation (E).The bars in A, B and C represent 1 mm, 1.5 mm, and 15mm and in D and E represent 0.5 mm.About 6 weeks after A. rhizogenes infection, we excised the hairy roots from the necrotic explant tissues and subcultured them on fresh agar-solidified selection medium. Mature hairy roots generally became thicker following subculture. After repeated transfer to fresh selection medium for 2 to 3 months, rapidly growing hairy root cultures of peanut were transferred to liquid culture medium containing 50 mg/L kanamycin and 250 mg/L Timentin (Figure 1-C).Next, we tested for integration of the NPTII gene into the genome of the peanut hairy root cultures. Then, we performed histochemical localization of GUS activity in various tissues, and checked for the presence of GUS mRNA, and the level of GUS enzyme activity. PCR experiments, which used primers specific for sequences in the NPTПgene, resulted in the amplification of a single amplicon with the expected size of 823 bp in 16 of 20 (data not shown). We then performed cytohistochemical staining for GUS enzyme activity to confirm that we had created completely transgenic peanut hairy roots and not chimeric cells that had transgenic and wild type tissues. The cauliflower mosaic virus (CaMV) 35S promoter-GUS fusion gene contained in the pBI 121 binary vector should result in constitutive GUS activity in all cells of transformed tissues. Indeed, we found strong GUS activity in the peanut hairy root vascular tissues of NPTП-positive cells after infection by A. rhizogenes strain R1000 with pBI 121 (Fig. 1-E). There was no GUS activity in any of the wild type peanut hairy root (Fig. 1-D).ABars represent the mean ± SD of three independent measurements (B).JONG SE KIM, SOOK YOUNG LEE, SANG UN PARKFollowing these results, we examined five randomly selected NPTП-positive hairy root lines to confirm the presence of GUS mRNA. Northern blots revealed high levels of GUS transcripts in each of the five hairy root lines (Fig. 2-A), but not in peanut wild type roots. These five hairy root lines also had much higher GUS enzyme activity than non-transformed roots (Fig. 2-B). There was some variation in GUS activity among the hairy root lines (from 776 to 1315 MU min-1mg-1protein), which is to be expected since there are likely to be differences in the five hairy root lines in the location of chromosomal insertion, the number of transgene copies, and in other post-translational effects.DiscussionsIn vitro production of resveratrol from callus, cell, and hairy root cultures of A. hypogaea has been reported [19; 14; 17; 11]. However, we describe here for the first time an efficient A. rhizogenes R1000 mediated transformation protocol for the establishment of transgenic peanut hairy root cultures. Genetically transformed hairy root cultures are ideal for study of the regulation of metabolic pathways that produce resveratrol and other important natural products. With our development of transformed peanut hairy root cultures, we expect to be able to more easily study the metabolic regulation of resveratrol in this species. AcknowledgmentsThis study was supported by research funds from Chosun University, 2008. References1. AGGARWAL B.B., BHARDWAJ A., AGGARWAL R.S., SEERAM N.P., SHISHODIA S., TAKADA Y.,(2004). Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 24: 2783-2840.2. BAUR J.A., PEARSON K.J., PRICE N.L., JAMIESON H.A., LERIN C., KALRA A., PRABHU V.V.,ALLARD J.S., LOPEZ-LLUCH G., LEWIS K., PISTELL P.J., POOSALA S., BECKER K.G., BOSS O., GWINN D., WANG M., RAMASWAMY S., FISHBEIN K.W., SPENCER R.G., LAKATTA E.G., LE COUTEUR D., SHAW R.J., NAVAS P., PUIGSERVER P., INGRAM D.K., DE CABO R., SINCLAIRD.A., (2006). Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444: 337-342.3. BRADFORD M.M. (1976). A rapid and sensitive method for quantization of microgram quantities of proteinusing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.4. BURNS J., YOKOTA T., ASHIHARA T., LEAN MEJ, CROZIER A., (2002). Plant foods and herbal sourcesof resveratrol. J. Agric. Food Chem. 50: 3337-3340.5. EDWARDS K., JOHNSTONE C., THOMPSON C., (1991). A simple and rapid method for preparation ofplant genomic DNA for PCR analysis. Nucleic Acids Research 19: 1349.6. FEINBERG A.P., VOGELSTEIN B.A., (1984). Technique for radiolabeling DNA restriction endonucleasefragments for high specific activity. Anal. Biochem. 137: 266-269.7. GIRI A., NARASU M.J., (2000). Transgenic hairy roots: recent trends and applications. BiotechnologyAdvances 18: 1-22.8. GUILLON S., TREMOUILLAUX-GUILLER J., PATI P.K., RIDEAU M., GANTET P., (2006). Harnessingthe potential of hairy roots: dawn of a new era. Trends Biotechnol. 24: 403-409.9. HAMILL J.D., PARR A.J., RHODES M.J.C., ROBINS R.J., WALTON N.J., (1987). New routes to plantsecondary products. Biotechnology 5: 800-804.10. JEFFERSON R.A., (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol.Rep. 5: 387-405.11. KIM J.S., LEE S.Y., PARK S.U., (2008). Resveratrol production in hairy root culture of peanut, Arachishypogaea L. transformed with different Agrobacterium rhizogenes strains. African Journal of Biotechnology. 7: 3785-3787.An efficient protocol for peanut (Arachis hypogaea L.) transformation mediated by Agrobacterium rhizogenes 12. KING R.E., KENT K.D., BOMSER J.A., (2005). Resveratrol reduces oxidation and proliferation of humanretinal pigment epithelial cells via extracellular signal-regulated kinase inhibition. Chem. Biol. Interact.151: 143-149.13. KOSUGI S., OHASHI Y., NAKAJIMA K., ARAI Y., (1990) An improved assay for β-glucuronidase intransformed cells: methanol almost completely suppresses a putative endogenous-glucuronidase activity.Plant Sci. 70: 133-140.14. KU K.L., CHANG P.S., CHENG Y.C., LIEN C.Y., (2005). Production of stilbenoids from the callus ofArachis hypogaea: a novel source of the anticancer compound piceatannol. J. Agric. Food Chem. 53: 3877-3881.15. LAUX M.T., AREGULLIN M., (2004). Identification of a p53-dependent pathway in the induction ofapoptosis of human breast cancer cells by the natural product, resveratrol. J. Altern. Complement Med. 10: 235-239.16. LOGEMANN J., SCHELL J., WILLMITZER L., (1987). Improved method for the isolation of RNA fromplant tissues. Anal. Biochem. 163: 16-20..17. MEDINA-BOLIVAR F., CONDORI J., RIMANDO A.M., HUBSTENBERGER J., SHELTON K.,O'KEEFE S.F., BENNETT S., DOLAN M.C., (2007). Production and secretion of resveratrol in hairy root cultures of peanut. Phytochemistry 68: 1992-2003.18. MURASHIGE T., SKOOG F., (1962) A revised medium for rapid growth and bioassays with tobacco tissuecultures. Physiol. Plant. 15: 473-497.19. ROLFS C.H., SCHÖN H., STEFFENS M., KINDL H. (1987).Cell-suspension culture of Arachis hypogaeaL.: model system of specific enzyme induction in secondary metabolism. Planta 172: 238-244.20. SAMBROOK J., FRITSCH E.F., MANIATIS T., (1989). Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY22. SHARMA K.K., BHATNAGAR-MATHUR P., (2006). Peanut (Arachis hypogaea L.). Methods Mol. Biol.343: 347-358.23. SIGNS M., FLORES H.,(1990). The biosynthetic potential of plant roots. Bioessays 12: 7-13.24. WENZEL E., SOMOZA V., (2005). Metabolism and bioavailability of trans-resveratrol. Mol. Nut. FoodRes. 49: 472-481.。

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不同处理方式对花生蛋白致敏性的影响

不同处理方式对花生蛋白致敏性的影响

圆园19年2月第40卷第3期食品研究与开发F ood Research And DevelopmentDOI ：10.3969/j.issn.1005-6521.2019.03.037作者简介：王闽佳（1994—），女（汉），在读研究生，研究方向：细胞生物学。

*通信作者：陈沁（1970—），女（汉），教授，博士，研究方向：生物化学与分子生物学。

不同处理方式对花生蛋白致敏性的影响王闽佳，丁岩汀，陈嘉惠，邓志瑞，陈沁*（上海大学生命科学学院，上海200444）摘要：花生蛋白是主要食物过敏原之一，会引起严重的甚至致命的食物过敏。

不同的加工处理方式都可以显著改变花生过敏原的分子结构，从而影响它们的致敏性。

结合已有的花生致敏的研究成果和最新的研究进展，分析比较花生传统热处理如烘烤处理、水煮处理、油炸处理和新型非热处理如超高压微射流处理、辐射处理、酶法处理对花生致敏性的影响，为加工生产低致敏性或无致敏性的花生制品提供理论依据。

关键词：热处理；非热处理；花生过敏原；蛋白结构；致敏性Influence of Different Treatments on the Allergenicity of Peanut ProteinWANG Min-jia ，DING Yan-ting ，CHEN Jia-hui ，DENG Zhi-rui ，CHEN Qin *（College of Life Sciences ，Shanghai University ，Shanghai 200444，China ）Abstract ：Peanut protein is one of the major food allergens ，which can cause severe or even fatal food allergies.Different processing methods can significantly alter the molecular structure of peanut allergens ，thus affecting their allergenicity.The existing research results and the latest research progress of peanut allergy were described.Besides ，the effects of traditional heat treatment of peanuts such as baking ，boiling ，frying and novelnon -thermal treatment such as ultra -high pressure micro -jet processing ，radiation treatment ，and enzyme treatment on peanut allergies ，were analyzed and compared.It provided a theoretical basis for the production ofpeanut products with low or no allergenicity.Key words ：heat treatment ；non-thermal treatment ；peanut allergen ；protein structure ；allergenicity引文格式：王闽佳，丁岩汀，陈嘉惠，等.不同处理方式对花生蛋白致敏性的影响[J].食品研究与开发，2019，40（3）：206-212WANG Minjia ，DING Yanting ，CHEN Jiahui ，et al.Influence of Different Treatments on the Allergenicity of Peanut Protein[J].Food Research and Development ，2019，40（3）：206-212过敏反应是指易过敏体质人群接触到周围环境中一些对健康体质无影响或影响微弱的过敏原因子，引起一系列严重的免疫反应和超敏反应的现象，如粉尘过敏和食物过敏。

花生及其产品介绍英文作文

花生及其产品介绍英文作文

花生及其产品介绍英文作文Title: Introduction to Peanuts and its Products。

Peanuts, scientifically known as Arachis hypogaea, are a versatile and nutritious legume that is widely consumed and appreciated around the globe. Originating from South America, peanuts have become a staple crop in many regions due to their rich flavor, high nutritional value, and diverse applications in culinary and industrial settings. In this essay, we will delve into the various aspects of peanuts and their products.1. Botanical Overview:Peanuts belong to the Fabaceae family and are classified as a legume. They grow underground, unlike most other legumes, which grow above ground. The peanut plant produces yellow flowers, which, after self-pollination, develop into pods containing the peanut seeds.2. Nutritional Value:Peanuts are a powerhouse of essential nutrients. They are rich in protein, healthy fats, fiber, vitamins, and minerals. A 100-gram serving of peanuts provides approximately 25 grams of protein, making them an excellent plant-based protein source for vegetarians and vegans. Additionally, peanuts contain monounsaturated and polyunsaturated fats, which are beneficial for heart health. They are also a good source of folate, niacin, vitamin E, and several minerals, including magnesium, phosphorus, and potassium.3. Culinary Uses:Peanuts are incredibly versatile in the kitchen and can be enjoyed in various forms. They are commonly consumed roasted or boiled as a snack. Peanut butter, a creamyspread made from ground peanuts, is a beloved condimentused in sandwiches, desserts, and savory dishes. Peanut oil, extracted from peanuts, is widely used in cooking due toits high smoke point and mild flavor. In Asian cuisines,peanuts are often used in stir-fries, sauces, and noodle dishes, adding texture and flavor to the recipes.4. Industrial Applications:Peanuts and their by-products have numerous industrial applications. Peanut oil serves as a key ingredient in cosmetics, soaps, and pharmaceuticals due to its moisturizing and emollient properties. Peanut shells, the outer covering of peanuts, are utilized in the production of animal feed, biomass fuel, and compost. Furthermore, peanut meal, a by-product of oil extraction, is used as a protein-rich supplement in livestock feed.5. Health Benefits:Including peanuts in your diet offers various health benefits. The high protein content of peanuts helps in muscle repair and growth, making them an excellent post-workout snack. The monounsaturated fats present in peanuts contribute to lowering bad cholesterol levels, thus reducing the risk of heart disease. Peanuts also containantioxidants such as resveratrol, which have anti-inflammatory properties and may reduce the risk of chronic diseases like cancer and diabetes when consumed as part of a balanced diet.6. Environmental Impact:The cultivation of peanuts can have both positive and negative environmental effects. On one hand, peanuts are nitrogen-fixing plants, which means they improve soil fertility by converting atmospheric nitrogen into a form that plants can use. This reduces the need for synthetic fertilizers, thus minimizing environmental pollution. However, large-scale peanut farming can lead to deforestation, habitat loss, and soil erosion if not managed sustainably. Therefore, it is essential for peanut producers to adopt practices that prioritize soil conservation and biodiversity preservation.In conclusion, peanuts are a versatile legume with a wide range of culinary, nutritional, and industrial applications. Whether enjoyed as a snack, incorporated intorecipes, or used in various industrial processes, peanuts continue to be valued for their flavor, nutrition, and utility. Moreover, as awareness of their health benefits and environmental impact grows, efforts to promote sustainable peanut production and consumption are becoming increasingly important.。

花生油的执行标准（Peanutoilstandards）

花生油的执行标准（Peanutoilstandards）

花生油的执行标准（Peanut oil standards）GB 1534-2003PrefaceTable of this standard 5.2, table 2, part indicators of Table 3, chapters 5.4 and seventh, and eighth are mandatory, while others are recommended.This standard is for GB1534-1986 "peanut oil", GB/T8615-19888, "sweet peanut oil" revision and merger.This standard is with GB1534-1986, GB/T 8615-19888 major technical differences:- the structure, technical elements and presentation rules of this standard shall be based on GB/T 1.1-200(00(guidelines for Standardization: first part: StandardStructure and writing rules;According to the processing method of peanut oil, classification and determination are carried out;- to adjust the standards and quality indicators in the above standards;- revised the relevant index value in the quality index.This standard has been revised in accordance with the international Codex Alimentarius Commission standards.This standard since the date of implementation, instead of GB1534-1986 "peanut oil", GB/T8615-1988 ((aromatic peanut oil).This standard is put forward by National Grain Bureau and put in order.This standard is responsible for drafting units: National Grain Bureau standard quality center, State Food Bureau, Xi'an food and feed quality supervision and inspectionTest center; in the drafting unit: Shandong Laiyang Luhua peanut oil Co. Ltd., Qingdao Kerry Vegetable Oil Co. Ltd. and Shanghai Fulinmen Food Co.Ltd, Shenzhen Nanshun Oil Co. ltd..The main drafters of this standard are Tang Ruiming, dragon, Xue Yalin, Chen Yan, Sun Dongwei, Pang Dongmei, Xu Xia, Xia Hongwen.All previous editions of the standard replaced by this standard are: GB1534-1986, GB/T8615-1988,GB 1534-2003Peanut oilRangeThis standard specifies the terms and definitions, classification, quality requirements, testing methods and rules, specifications, labels, packaging, storage and transportation of peanut oilRequirement。

《生物多样性公约》英文版

《生物多样性公约》英文版

CARTAGENA PROTOCOL ON BIOSAFETY TO THE CONVENTION ONBIOLOGICAL DIVERSITYThe Parties to this Protocol,Being Parties to the Convention on Biological Diversity, hereinafter referred to as "the Convention",Recalling Article 19, paragraphs 3 and 4, and Articles 8 (g) and 17 of the Convention,Recalling also decision II/5 of 17 November 1995 of the Conference of the Parties to the Convention to develop a Protocol on biosafety, specifically focusing on transboundary movement of any living modified organism resulting from modern biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity, setting out for consideration, in particular, appropriate procedures for advance informed agreement,Reaffirming the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development,Aware of the rapid expansion of modern biotechnology and the growing public concern over its potential adverse effects on biological diversity, taking also into account risksto human health,Recognizing that modern biotechnology has great potential for human well-being if developed and used with adequate safety measures for the environment and human health,Recognizing also the crucial importance to humankind of centres of origin and centres of genetic diversity,Taking into account the limited capabilities of many countries, particularly developing countries, to cope with the nature and scale of known and potential risks associated with living modified organisms,Recognizing that trade and environment agreements should be mutually supportive with a view to achieving sustainable development,Emphasizing that this Protocol shall not be interpreted as implying a change in the rights and obligations of a Party under any existing international agreements,Understanding that the above recital is not intended to subordinate this Protocol to other international agreements,Have agreed as follows:Article 1OBJECTIVEIn accordance with the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development, the objective of this Protocol is to contribute to ensuring an adequate level of protection in the field of the safe transfer, handling and use of living modified organisms resulting from modern biotechnology that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, and specifically focusing on transboundary movements.Article 2GENERAL PROVISIONS1. Each Party shall take necessary and appropriate legal, administrative and other measures to implement its obligations under this Protocol.2. The Parties shall ensure that the development, handling, transport, use, transfer and release of any living modified organisms are undertaken in a manner that prevents or reduces the risks to biological diversity, taking also into account risks to human health.3. Nothing in this Protocol shall affect in any way the sovereignty of States over their territorial sea established in accordance with international law, and the sovereign rights and the jurisdiction which States have in their exclusive economic zones and their continental shelves in accordance with international law, and the exercise by ships and aircraft of all States of navigational rights and freedoms as provided for in international law and as reflected in relevant international instruments.4. Nothing in this Protocol shall be interpreted as restricting the right of a Party to take action that is more protective of the conservation and sustainable use of biological diversity than that called for in this Protocol, provided that such action is consistentwith the objective and the provisions of this Protocol and is in accordance with thatParty's other obligations under international law.5. The Parties are encouraged to take into account, as appropriate, available expertise, instruments and work undertaken in international forums with competence in the area of risks to human health.Article 3USE OF TERMSFor the purposes of this Protocol:(a) "Conference of the Parties" means the Conference of the Parties to the Convention;(b) "Contained use" means any operation, undertaken within a facility,installation or other physical structure, which involves living modified organisms that are controlled by specific measures that effectively limit their contact with, and their impact on, the external environment;(c) "Export" means intentional transboundary movement from one Party to another Party;(d) "Exporter" means any legal or natural person, under the jurisdiction of the Party of export, who arranges for a living modified organism to be exported;(e) "Import" means intentional transboundary movement into one Party from another Party;(f) "Importer" means any legal or natural person, under the jurisdiction of the Party of import, who arranges for a living modified organism to be imported;(g) "Living modified organism" means any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology;(h) "Living organism" means any biological entity capable of transferring or replicating genetic material, including sterile organisms, viruses and viroids;(i) "Modern biotechnology" means the application of:a. In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid(DNA) and direct injection of nucleic acid into cells or organelles, orb. Fusion of cells beyond the taxonomic family,that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection;(j) "Regional economic integration organization" means an organization constituted by sovereign States of a given region, to which its member States have transferred competence in respect of matters governed by this Protocol and which has been duly authorized, in accordance with its internal procedures, to sign, ratify, accept, approve or accede to it;(k) "Transboundary movement" means the movement of a living modified organism from one Party to another Party, save that for the purposes of Articles 17 and 24 transboundary movement extends to movement between Parties and non-Parties.Article 4SCOPEThis Protocol shall apply to the transboundary movement, transit, handling and use of all living modified organisms that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health.Article 5PHARMACEUTICALSNotwithstanding Article 4 and without prejudice to any right of a Party to subjectall living modified organisms to risk assessment prior to the making of decisions on import, this Protocol shall not apply to the transboundary movement of living modified organisms which are pharmaceuticals for humans that are addressed by other relevant international agreements or organisations.Article 6TRANSIT AND CONTAINED USE1. Notwithstanding Article 4 and without prejudice to any right of a Party of transit to regulate the transport of living modified organisms through its territory and make available to the Biosafety Clearing-House, any decision of that Party, subject to Article 2, paragraph 3, regarding the transit through its territory of a specific living modified organism, the provisions of this Protocol with respect to the advance informed agreement procedure shall not apply to living modified organisms in transit.2. Notwithstanding Article 4 and without prejudice to any right of a Party to subjectall living modified organisms to risk assessment prior to decisions on import and to set standards for contained use within its jurisdiction, the provisions of this Protocol with respect to the advance informed agreement procedure shall not apply to the transboundary movement of living modified organisms destined for contained use undertaken in accordance with the standards of the Party of import.Article 7APPLICATION OF THE ADVANCE INFORMED AGREEMENT PROCEDURE1. Subject to Articles 5 and 6, the advance informed agreement procedure in Articles 8 to 10 and 12 shall apply prior to the first intentional transboundary movement of living modified organisms for intentional introduction into the environment of the Party of import.2. "Intentional introduction into the environment" in paragraph 1 above, does not refer to living modified organisms intended for direct use as food or feed, or for processing.3. Article 11 shall apply prior to the first transboundary movement of living modified organisms intended for direct use as food or feed, or for processing.4. The advance informed agreement procedure shall not apply to the intentional transboundary movement of living modified organisms identified in a decision of the Conference of the Parties serving as the meeting of the Parties to this Protocol as being not likely to have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health.Article 8NOTIFICATION1. The Party of export shall notify, or require the exporter to ensure notification to, in writing, the competent national authority of the Party of import prior to theintentional transboundary movement of a living modified organism that falls within the scope of Article 7, paragraph 1. The notification shall contain, at a minimum, the information specified in Annex I.2. The Party of export shall ensure that there is a legal requirement for the accuracy of information provided by the exporter.Article 9ACKNOWLEDGEMENT OF RECEIPT OF NOTIFICATION1. The Party of import shall acknowledge receipt of the notification, in writing, to the notifier within ninety days of its receipt.2. The acknowledgement shall state:(a) The date of receipt of the notification;(b) Whether the notification, prima facie, contains the information referred to in Article 8;(c) Whether to proceed according to the domestic regulatory framework of the Party of import or according to the procedure specified in Article 10.3. The domestic regulatory framework referred to in paragraph 2 (c) above, shall be consistent with this Protocol.4. A failure by the Party of import to acknowledge receipt of a notification shall not imply its consent to an intentional transboundary movement.Article 10DECISION PROCEDURE1. Decisions taken by the Party of import shall be in accordance with Article 15.2. The Party of import shall, within the period of time referred to in Article 9, inform the notifier, in writing, whether the intentional transboundary movement may proceed:(a) Only after the Party of import has given its written consent; or(b) After no less than ninety days without a subsequent written consent.3. Within two hundred and seventy days of the date of receipt of notification, the Party of import shall communicate, in writing, to the notifier and to the Biosafety Clearing-House the decision referred to in paragraph 2 (a) above:(a) Approving the import, with or without conditions, including how the decision will apply to subsequent imports of the same living modified organism;(b) Prohibiting the import;(c) Requesting additional relevant information in accordance with its domestic regulatory framework or Annex I; in calculating the time within which the Party of importis to respond, the number of days it has to wait for additional relevant information shall not be taken into account; or(d) Informing the notifier that the period specified in this paragraph is extended by a defined period of time.4. Except in a case in which consent is unconditional, a decision under paragraph 3 above, shall set out the reasons on which it is based.5. A failure by the Party of import to communicate its decision within two hundred and seventy days of the date of receipt of the notification shall not imply its consent to an intentional transboundary movement.6. Lack of scientific certainty due to insufficient relevant scientific information and knowledge regarding the extent of the potential adverse effects of a living modified organism on the conservation and sustainable use of biological diversity in the Party of import, taking also into account risks to human health, shall not prevent that Party from taking a decision, as appropriate, with regard to the import of the living modified organism in question as referred to in paragraph 3 above, in order to avoid or minimize suchpotential adverse effects.7. The Conference of the Parties serving as the meeting of the Parties shall, at itsfirst meeting, decide upon appropriate procedures and mechanisms to facilitate decision-making by Parties of import.Article 11PROCEDURE FOR LIVING MODIFIED ORGANISMS INTENDED FOR DIRECT USE AS FOOD OR FEED, OR FORPROCESSING1. A Party that makes a final decision regarding domestic use, including placing on the market, of a living modified organism that may be subject to transboundary movement for direct use as food or feed, or for processing shall, within fifteen days of making that decision, inform the Parties through the Biosafety Clearing-House. This information shall contain, at a minimum, the information specified in Annex II. The Party shall provide a copy of the information, in writing, to the national focal point of each Party that informs the Secretariat in advance that it does not have access to the Biosafety Clearing-House. This provision shall not apply to decisions regarding field trials.2. The Party making a decision under paragraph 1 above, shall ensure that there is a legal requirement for the accuracy of information provided by the applicant.3. Any Party may request additional information from the authority identified in paragraph (b) of Annex II.4. A Party may take a decision on the import of living modified organisms intended for direct use as food or feed, or for processing, under its domestic regulatory framework that is consistent with the objective of this Protocol.5. Each Party shall make available to the Biosafety Clearing-House copies of anynational laws, regulations and guidelines applicable to the import of living modified organisms intended for direct use as food or feed, or for processing, if available.6. A developing country Party or a Party with an economy in transition may, in the absence of the domestic regulatory framework referred to in paragraph 4 above, and in exercise of its domestic jurisdiction, declare through the Biosafety Clearing-House that its decision prior to the first import of a living modified organism intended for direct use as food or feed, or for processing, on which information has been provided under paragraph 1 above, will be taken according to the following:(a) A risk assessment undertaken in accordance with Annex III; and(b) A decision made within a predictable timeframe, not exceeding two hundred and seventy days.7. Failure by a Party to communicate its decision according to paragraph 6 above, shall not imply its consent or refusal to the import of a living modified organism intended for direct use as food or feed, or for processing, unless otherwise specified by the Party.8. Lack of scientific certainty due to insufficient relevant scientific information and knowledge regarding the extent of the potential adverse effects of a living modified organism on the conservation and sustainable use of biological diversity in the Party of import, taking also into account risks to human health, shall not prevent that Party from taking a decision, as appropriate, with regard to the import of that living modifiedorganism intended for direct use as food or feed, or for processing, in order to avoid or minimize such potential adverse effects.9. A Party may indicate its needs for financial and technical assistance and capacity-building with respect to living modified organisms intended for direct use as food or feed, or for processing. Parties shall cooperate to meet these needs in accordance with Articles 22 and 28.Article 12REVIEW OF DECISIONS1. A Party of import may, at any time, in light of new scientific information on potential adverse effects on the conservation and sustainable use of biological diversity, taking also into account the risks to human health, review and change a decision regarding an intentional transboundary movement. In such case, the Party shall, within thirty days, inform any notifier that has previously notified movements of the living modified organism referred to in such decision, as well as the Biosafety Clearing-House, and shall set out the reasons for its decision.2. A Party of export or a notifier may request the Party of import to review a decision it has made in respect of it under Article 10 where the Party of export or the notifier considers that:(a) A change in circumstances has occurred that may influence the outcome of the risk assessment upon which the decision was based; or(b) Additional relevant scientific or technical information has become available.3. The Party of import shall respond in writing to such a request within ninety days and set out the reasons for its decision.4. The Party of import may, at its discretion, require a risk assessment for subsequent imports.Article 13SIMPLIFIED PROCEDURE1. A Party of import may, provided that adequate measures are applied to ensure the safe intentional transboundary movement of living modified organisms in accordance with the objective of this Protocol, specify in advance to the Biosafety Clearing-House:(a) Cases in which intentional transboundary movement to it may take place at the same time as the movement is notified to the Party of import; and(b) Imports of living modified organisms to it to be exempted from the advance informed agreement procedure.Notifications under subparagraph (a) above, may apply to subsequent similar movements to the same Party.2. The information relating to an intentional transboundary movement that is to be provided in the notifications referred to in paragraph 1 (a) above, shall be the information specified in Annex I.Article 14BILATERAL, REGIONAL AND MULTILATERAL AGREEMENTS AND ARRANGEMENTS1. Parties may enter into bilateral, regional and multilateral agreements and arrangements regarding intentional transboundary movements of living modified organisms, consistent with the objective of this Protocol and provided that such agreements and arrangements do not result in a lower level of protection than that provided for by the Protocol.2. The Parties shall inform each other, through the Biosafety Clearing-House, of any such bilateral, regional and multilateral agreements and arrangements that they have entered into before or after the date of entry into force of this Protocol.3. The provisions of this Protocol shall not affect intentional transboundary movements that take place pursuant to such agreements and arrangements as between the parties to those agreements or arrangements.4. Any Party may determine that its domestic regulations shall apply with respect to specific imports to it and shall notify the Biosafety Clearing-House of its decision.Article 15RISK ASSESSMENT1. Risk assessments undertaken pursuant to this Protocol shall be carried out in a scientifically sound manner, in accordance with Annex III and taking into account recognized risk assessment techniques. Such risk assessments shall be based, at a minimum, on information provided in accordance with Article 8 and other available scientific evidence in order to identify and evaluate the possible adverse effects of living modified organisms on the conservation and sustainable use of biological diversity, taking also into account risks to human health.2. The Party of import shall ensure that risk assessments are carried out for decisions taken under Article 10. It may require the exporter to carry out the risk assessment.3. The cost of risk assessment shall be borne by the notifier if the Party of import so requires.Article 16RISK MANAGEMENT1. The Parties shall, taking into account Article 8 (g) of the Convention, establish and maintain appropriate mechanisms, measures and strategies to regulate, manage and control risks identified in the risk assessment provisions of this Protocol associated with the use, handling and transboundary movement of living modified organisms.2. Measures based on risk assessment shall be imposed to the extent necessary to prevent adverse effects of the living modified organism on the conservation and sustainable use ofbiological diversity, taking also into account risks to human health, within the territory of the Party of import.3. Each Party shall take appropriate measures to prevent unintentional transboundary movements of living modified organisms, including such measures as requiring a risk assessment to be carried out prior to the first release of a living modified organism.4. Without prejudice to paragraph 2 above, each Party shall endeavour to ensure that any living modified organism, whether imported or locally developed, has undergone an appropriate period of observation that is commensurate with its life-cycle or generation time before it is put to its intended use.5. Parties shall cooperate with a view to:(a) Identifying living modified organisms or specific traits of living modified organisms that may have adverse effects on the conservation and sustainable use ofbiological diversity, taking also into account risks to human health; and(b) Taking appropriate measures regarding the treatment of such living modified organisms or specific traits.Article 17UNINTENTIONAL TRANSBOUNDARY MOVEMENTS AND EMERGENCY MEASURES1. Each Party shall take appropriate measures to notify affected or potentially affected States, the Biosafety Clearing-House and, where appropriate, relevant international organizations, when it knows of an occurrence under its jurisdiction resulting in a release that leads, or may lead, to an unintentional transboundary movement of a living modified organism that is likely to have significant adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health in such States. The notification shall be provided as soon as the Party knows of the above situation.2. Each Party shall, no later than the date of entry into force of this Protocol for it, make available to the Biosafety Clearing-House the relevant details setting out its point of contact for the purposes of receiving notifications under this Article.3. Any notification arising from paragraph 1 above, should include:(a) Available relevant information on the estimated quantities and relevant characteristics and/or traits of the living modified organism;(b) Information on the circumstances and estimated date of the release, and on the use of the living modified organism in the originating Party;(c) Any available information about the possible adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, as well as available information about possible risk management measures;(d) Any other relevant information; and(e) A point of contact for further information.4. In order to minimize any significant adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, each Party, under whose jurisdiction the release of the living modified organism referred to in paragraph 1 above, occurs, shall immediately consult the affected or potentiallyaffected States to enable them to determine appropriate responses and initiate necessary action, including emergency measures.Article 18HANDLING, TRANSPORT, PACKAGING AND IDENTIFICATION1. In order to avoid adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, each Party shall take necessary measures to require that living modified organisms that are subject to intentional transboundary movement within the scope of this Protocol are handled, packaged and transported under conditions of safety, taking into consideration relevant international rules and standards.2. Each Party shall take measures to require that documentation accompanying:(a) Living modified organisms that are intended for direct use as food or feed, or for processing, clearly identifies that they "may contain" living modified organisms and are not intended for intentional introduction into the environment, as well as a contact point for further information. The Conference of the Parties serving as the meeting of theParties to this Protocol shall take a decision on the detailed requirements for this purpose, including specification of their identity and any unique identification, no later than two years after the date of entry into force of this Protocol;(b) Living modified organisms that are destined for contained use clearlyidentifies them as living modified organisms; and specifies any requirements for the safe handling, storage, transport and use, the contact point for further information, including the name and address of the individual and institution to whom the living modified organisms are consigned; and(c) Living modified organisms that are intended for intentional introduction into the environment of the Party of import and any other living modified organisms within the scope of the Protocol, clearly identifies them as living modified organisms; specifies the identity and relevant traits and/or characteristics, any requirements for the safe handling, storage, transport and use, the contact point for further information and, as appropriate, the name and address of the importer and exporter; and contains a declaration that the movement is in conformity with the requirements of this Protocol applicable to the exporter.3. The Conference of the Parties serving as the meeting of the Parties to this Protocol shall consider the need for and modalities of developing standards with regard to identification, handling, packaging and transport practices, in consultation with other relevant international bodies.Article 19COMPETENT NATIONAL AUTHORITIES AND NATIONAL FOCAL POINTS1. Each Party shall designate one national focal point to be responsible on its behalf for liaison with the Secretariat. Each Party shall also designate one or more competentnational authorities, which shall be responsible for performing the administrative functions required by this Protocol and which shall be authorized to act on its behalf with respect to those functions. A Party may designate a single entity to fulfil the functions of both focal point and competent national authority.2. Each Party shall, no later than the date of entry into force of this Protocol for it, notify the Secretariat of the names and addresses of its focal point and its competent national authority or authorities. Where a Party designates more than one competent national authority, it shall convey to the Secretariat, with its notification thereof, relevant information on the respective responsibilities of those authorities. Where applicable, such information shall, at a minimum, specify which competent authority is responsible for which type of living modified organism. Each Party shall forthwith notify the Secretariat of any changes in the designation of its national focal point or in the name and address or responsibilities of its competent national authority or authorities.3. The Secretariat shall forthwith inform the Parties of the notifications it receives under paragraph 2 above, and shall also make such information available through the Biosafety Clearing-House.Article 20INFORMATION SHARING AND THE BIOSAFETY CLEARING-HOUSE1. A Biosafety Clearing-House is hereby established as part of the clearing-house mechanism under Article 18, paragraph 3, of the Convention, in order to:(a) Facilitate the exchange of scientific, technical, environmental and legal information on, and experience with, living modified organisms; and(b) Assist Parties to implement the Protocol, taking into account the special needs of developing country Parties, in particular the least developed and small island developing States among them, and countries with economies in transition as well ascountries that are centres of origin and centres of genetic diversity.2. The Biosafety Clearing-House shall serve as a means through which information is made available for the purposes of paragraph 1 above. It shall provide access to information。