Plant Surface Sterilization Kit
Contents
1. of the liquid can be decanted into a waste container but use caution that the sample is not lost 6 Add enough Sterilization Solution B to submerge the sample For most seed or explant samples the volume will be approximately 5 ml If using a microcentrifuge tube a volume of 500 ul should be sufficient 7 Soak seeds or explant in Sterilization Solution B for 15 minutes with occasional agitation The closed container with the sample may be placed on a shaking platform with gentle rocking or may be swirled or inverted by hand every few minutes 8 Remove as much of the residual liquid as possible using a sterile pipet The majority of the liquid can be decanted into a waste container but use caution that the sample is not lost 9 Add enough sterile distilled water to submerge the sample For most seed or explant samples the volume will be approximately 5 ml If using a microcentrifuge tube a volume of 500 ul should be sufficient 10 Incubate at room temperature for 5 minutes with occasional agitation The closed container with the sample may be placed on a shaking platform with gentle rocking or may be swirled or inverted by hand every few minutes 11 Remove as much of the residual liquid as possible using a sterile pipet Repeat steps 9 11 two more times The sample is now ready for in vitro culture 12 Flame sterilize or autoclave the forceps provided in the kit 13 Using the cooled sterile forceps transfer the seeds or exp2. Plant Surface Sterilization Kit Rapid Sterilization of Plant Seeds and Tissues for in vitro Growth Revision 5100 200 3G01 Surface Sterilization Kit Plant Surface Sterilization Kit Rapid Sterilization of Plant Seeds and Tissues for in vitro Growth Application Manual Revision 5100 200 3G01 Catalog 5100 200 50 Samples Storage temperature Ambient temperature 15 30 C Any Questions Call Technical Support at 800 424 6101 3 Plant Surface Sterilizatio TABLE OF CONTENTS 1 Introduction sau 5 2 Kit Components and User Supplied Materials 5 2 1 Plant Surface Sterilization Kit Components 5 2 2 User het aid odia 5 3 Safety PRECAUTIONS iii aaa 6 4 Detailed Protocol for Seed and Explant Sterilization 6 5 5 8 5 1 No Germination or Slow 8 5 2 Bacterial and Fungal Contamination 8 6 Recommended Reference Format for Publication 9 7 References i2 ce nn eR a A ae 9 8 Related Productsia a ei 10 9 Product Use Limitation amp Watranty cian 11 4 visit us on the web at www qbiogene com Surface Sterilization Kit 1 Introduction Plant tissue culture also known as in vitro plant culture or micro propag
3. ation is the growth or maintenance of plant cells tissues or whole plants in vitro using a defined growth medium Plastic and glass vessels are used for solid and liquid culture to maintain plant cells seeds or tissues in a sterile environment The Plant Surface Sterilization Kit is designed to sterilize seeds for germination and plant tissues for callus induction without affecting the ability of the seed or tissue to grow in culture or at a later time in soil The two solutions in this kit preserve plant tissue characteristics for successful growth while providing surface sterilization to remove contaminating microorganisms associated with plant seeds and plant tissues explants prior to in vitro incubation using sterile growth medium The following protocol has been developed as a standard guideline for surface sterilization and will work on a wide variety of seed and explant types Specific seeds and tissues may require modification to this basic pro tocol For example larger plant seed and tissue samples often require longer sterilization time Storage plant tissues tubers may need to be peeled and cored before sterilization and prior to plating on culture media 2 Kit Components and User Supplied Materials 2 1 Plant Surface Sterilization Kit Components Description Quantity Sterilization Solution A 1 x 250 ml bottle Sterilization Solution B 1 x 250 ml bottle Reusable Polypropylene Container for Large Plant Samples 1 each Reusabl
4. cription amp Size Nucleic Acid Purification Products 6045 050 FastRNA Pro Green Kit Plants and Animals 50 preps 6540 400 FastDNA Kit 100 preps 6560 200 FastDNA SPIN Kit for Soil 50 preps 6001 100 FP100A Instrument 100V 6001 120 FP120A Instrument 120V 6001 220 FP220A Instrument 220V 6025 050 FastRNA Pro Blue Kit Bacteria 50 preps 6035 050 FastRNA Pro Red Kit Yeast and Fungi 50 preps 2012 400 Floraclean M Kit 25 preps 9 Product Use Limitation amp Warranty Unless otherwise indicated this product is for research use only Purchase of Qbiogene Inc products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties Qbiogene Inc makes no warranty of any kind expressed or implied including merchantability or fitness for any particular purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liability of Qbiogene Inc hereunder shall be limited to at our dis cretion no replacement or compensation product credits refund of the purchase price of or the replace ment of materials that do not meet our specification By acceptance of the product Buyer indemnifies and holds Qbiogene Inc harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but n
5. e Polypropylene Container for Small Plant Samples 1 each Dissecting Forceps 1 each User Manual 1 each MSDS 1 each Certificate of Analysis 1 each 2 2 User Supplied Materials Plant Tissue Culture Medium See Section 8 Related Products Appropriate Tissue Culture Plates or Boxes see Section 8 Related Products Appropriate Plant Growth Regulators Sterile Deionized or Distilled Water autoclaved or 0 2 um filtered Sterile Environment laminar flow culture hood Any Questions Call Technical Support at 800 424 6101 5 Plant Surface Sterilizat 3 Safety Precautions Sterilization Solutions A and B contain components that may cause irritation or burning when in contact with human tissue or during inhalation Wear personal protective equipment to prevent skin contact e g gloves lab coat and eye protection and prevent inhalation of reagent vapors and consumption of liquid during use Consult the enclosed Material Safety Data Sheet for additional details 4 Detailed Protocol for Seed and Explant Sterilization NOTE Recommended incubation times are used for the majority of applications and may be adapted to meet individual requirements NOTE The size and specific type of explant tissue necessary for in vitro culture varies by plant and by overall experimental procedure and is not described in this protocol 1 If seeds or explant contains obvious dirt or other contaminants remove them by rinsing with water NOTE f necessar
6. l Liquid 5101 035 Gamborg s B5 Basal Salts 10L Pouch Powder 5101 014 Gamborg s B5 Basal Salts 10X Liquid 500ml Liquid 5101 335 Gamborg s B5 Basal Medium Salts Vitamins 10L Pouch Powder 5101 125 Gamborg s B5 Complete Medium Salts Vitamins Sucrose 1L Pouch Powder 5101 114 Gamborg s B5 Complete Medium Salts Vitamins Sucrose liquid 500ml Liquid 5101 225 Gamborg s B5 Complete Agar Salts Vitamins Sucrose Agar 1L Pouch Powder 5101 042 Gamborg s Vitamins Powder 5g Powder 5101 044 Gamborg s Vitamins 1000X Liquid 20ml Liquid 5102 035 Chu s Basal Salts 10L Pouch Powder 5102 014 Chu s Basal Salts 10X Liquid 500ml Liquid 5102 335 Chu s Basal Medium Salts Vitamins 10L Pouch Powder 5102 125 Chu s Complete Medium Salts Vitamins Sucrose 1L Pouch Powder 5102 114 Chu s Complete Medium Salts Vitamins Sucrose Liquid 500ml Liquid 5102 225 Chu s Complete Agar Salts Vitamins Sucrose Agar 1L PouchPowder 5102 042 Chu s Vitamins Powder 5g Powder 5102 044 Chu s Vitamins 1000X Liquid 20ml Liquid Culture Dishes 5110 033 Magenta Box GA7 Pack of 8 Pack 5110 053 Magenta Box GA7 5 packs of 8 Pack 5110 043 Magenta Box Tray 7 Way Each 5111 033 Petri Dishes Sterile Polystyrene 100mm x 20mm 20 plates Sleeve 5111 053 Petri Dishes Sterile Polystyrene 100mm x 20mm 25 sleeves of 20 Sleeve 10 visit us on the web at www qbiogene com Surface Sterilization Kit Cat Des
7. lant tissue to an agar plate containing the appropriate growth media and growth regulators Small seeds may be transferred in the water using a sterile transfer pipet 14 Maintain cultures at approximately 25 C under low to moderate intensity light or follow plant specific growth requirements If possible incubate plates at an angle sufficient to cause excess water to drain away from the sample Any Questions Call Technical Support at 800 424 6101 7 Plant Surface Sterili 15 Depending on the type of seeds or explant germination will occur within approximately 1 to 4 weeks For example Alfalfa Arabidopsis and California poppy will germinate in approximately one week citrus will require approximately 2 4 weeks Callus initiation will typically appear after 2 to 4 weeks NOTE If inducing callus formation on the agar plate large sterilized seeds should be cut in half and large sterilized explants should be sliced prior to plating on culture media Sterile instruments must be used at all times NOTE The germination period for seeds or explants that have been stored may be longer than the time needed by fresh seeds Factors such as the sample age storage conditions type and pre harvest factors will contribute to the time required for germination and the overall success rate of the experiment Seed germination efficiency is typically less than 100 16 Examine cultures every 3 5 days for contamination Slick or shiny areas
8. oberts Experiments in Plant Tissue Culture 3rd Ed Cambridge University Press 1995 3 Gamborg O L R A Miller and Ojima 1968 Nutrient Requirements of Suspension Cultures of Soybean Root Cells Experimental Cell Research 50 151 158 4 Gamborg L T Murashige Thorpe and I Vasil 1976 Plant Tissue Culture Media In vitro 12 7 473 478 Murashige T and F Skoog 1962 A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures Physiologia Plantarum 15 473 497 5 Sharp W R D A Evans P V Ammirato and Y Yamada Eds Handbook of Plant Cell Culture 2 198 Macmillan Publishing Company 1984 Any Questions Call Technical Support at 800 424 6101 9 Plant Surface Sterili 8 Related Products Cat Description amp Size Format Growth Media 5100 035 Murashige amp Skoog Basal Salts 10L Pouch Powder 5100 014 Murashige amp Skoog Basal Salts 10X Liquid 500ml Liquid 5100 335 Murashige amp Skoog Basal Medium Salts Vitamins 10L Pouch Powder 5100 125 Murashige amp Skoog Complete Medium Salts Vitamins Sucrose 1L Pouch Powder 5100 114 Murashige amp Skoog Complete Medium Salts Vitamins Sucrose Liquid 500ml Liquid 5100 225 Murashige amp Skoog Complete Agar Salts Vitamins Sucrose Agar 1L Pouch Powder 5100 042 Murashige amp Skoog Vitamins Powder 5g Powder 5100 044 Murashige amp Skoog Vitamins 1000X Liquid 20m
9. on the agar are typically bacterial contamination while fuzzy areas are typically due to fungal contamination Do not open containers that are contaminated Seal lid and plate together using Parafilm or tape and decontaminate by autoclaving for at least 15 minutes at 15 psi Discard appropriately See Section 5 2 for troubleshooting contamination problems 5 Troubleshooting 5 1 Germination or Slow Germination Ensure that sterilized seed or tissue is rinsed thoroughly with sterile water prior to placing on growth media For small seeds Arabidopsis or Orchid or fragile organs anther stamen pistil the incubation time in Sterilization Solution B may be reduced from 15 minutes to 5 10 minutes Sixteen hours of fluorescent light and 8 hours of darkness are recommended as starting conditions for most plants Adjust light exposure as needed to optimize germination 5 2 Bacterial and Fungal Contamination Successful control of contamination depends largely upon strict adherence to aseptic techniques By observing where on a plate contamination arises it may be possible to determine its source For example contamination not associated with the sample suggests potential airborne or culture media sources Aseptic techniques include appropriate covering of hands and clothing and ensuring that nothing unnecessary moves near or disturbs open or partially uncovered samples or plates 8 visit us on the web at www qbiogene com Surface S
10. ot limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying Qbiogene Inc within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s Any Questions Call Technical Support at 800 424 6101 11 Plant Surface Sterilization Kit NOTES 12 visit us on the web at www gbiogene com Plant Surface Sterilization Kit NOTES Any Questions Call Technical Support at 800 424 6101 13 Plant Surface Sterilization Kit NOTES 14 visit us on the web at www gbiogene com
11. terilization Kit An approved laminar flow hood with ultraviolet decontamination is recommended for all sterilizing and cul turing activities Treating the work area with 70 ethanol 10 bleach or other sterilization solutions prior to use is recommended Additional decontamination preventive measures may also include sterilizing the work area floor with a decontaminating agent keeping doors and windows closed during the procedure and filtering 0 2 um air entering the work area A sterile work surface e g sterile Petri dish or culture plate is recommended as a surface on which to trim sterilized tissue prior to culturing Although antibiotics are used routinely in animal cell cultures they are not widely used in plant tissue cul tures because they are often ineffective kill the culture or induce chromosomal instability Plants may metabolize antibiotics or their degradation products with unpredictable results If antibiotic usage is deemed necessary the agents of choice are those that act specifically within bacterial cell walls and bacterial mem branes Selection of an effective agent cannot be made until the contaminants are known 6 Recommended Reference Format for Publications Specific seed or tissue explant was sterilized prior to in vitro culture using the Plant Surface Sterilization Kit Qbiogene Inc CA 7 References 1 Chu Z Q et al 1975 Chinese Academie Sinica 5 484 490 2 Dodds John and Lorin W R
12. y a mild soapy detergent may be used to remove dirt and or contaminants Thoroughly rinse seeds or explant with water to remove detergent before proceeding 2 Place the seeds or explant in the small or large sterilization container provided in the kit Choose the container which provides enough area for the sterilization solutions to cover the samples NOTE The Plant Surface Sterilization Kit is designed for batch processing seed and tissue sam ples A pouch or filter bag is not required to hold the sample but the solutions may be used with cheesecloth or other pouching methods if necessary When using a pouch ensure adequate con tact occurs between the sample and the sterilization solutions during the process NOTE For very small seeds or explants a smaller container such as a microcentrifuge tube can be used to contain the sample during processing 3 Add enough Sterilization Solution A to submerge the sample For most seed or explant samples the volume will be approximately 5 ml If using a microcentrifuge tube a volume of 500 ul should be sufficient 4 Incubate for 5 minutes at room temperature with occasional agitation The closed container with the sample may be placed on a shaking platform with gentle rocking or may be swirled or inverted by hand every few minutes 6 visit us on the web at www qbiogene com rface Sterilization Kit 5 Remove as much of the residual liquid as possible using a sterile pipet The majority
Download Pdf Manuals
Related Search