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This file describes the procedures that are presently being used in alfalfa tissue culture to produce dry somatic embryos that may be used as artificial seeds. Select one of the following topics to review the procedure in detail: Sterilization and Induction Suspension Culture Sieving The Suspension Development and Maturation Drying Germination References
Sterilization and Induction
1. Harvest petioles from shoots of donor plants. Petioles from young fully expanded leaves are the best; avoid selecting petioles from flowering shoots. 2. Immerse petioles in 70% ethanol for 30-40 seconds and then in 25% commercial bleach (25 ml bleach + 75 ml water) for 20 minutes. Rinse petioles with three generous changes of sterile water. 3. Place sterile petioles in Petri dish containing sterile water to keep the material hydrated until further processing. 4. Cut petioles into 5-10 mm lengths; 8 mm is best, but depending on the experiment this may vary. 5. Place 10 petioles on a Petri plate containing SH induction medium. 6. An alternative explant that produces good callus growth is an immature embryo at the torpedo stage of development. 7. Incubate the plates of induction medium at 25°C, 16 hour photoperiod, 75 µmol m2s-1 PPFD for 14 to 21 days. Callus at this stage should be friable; the petiole should be completely overgrown with callus. If the callus is hard or not healthy in
If the suspension is subcultured too frequently. Transfer petiole-derived callus from induction medium to 125 ml flasks containing 40 ml of B5 suspension medium. Click here to view picture of callus Go to next topic Return to Table of Contents Suspension Culture 1. Usually this will give another equivalent batch of embryos. veloping in suspension are larger thanzygotic embryos and lack a normal suspensor. This sometimes happens due to poor donor plant health. 16 hour photoperiod 30 µmoles m-2s-1 PPFD for a period of 7 to 14 days. Place flasks on shaker at 25C. Wash the 0. Discard callus left on 0. . at a rate of approximately 1 gram of petiolederived callus into each flask. the small single cells begin to dominate the culture and its ability to form somatic embryos is lost.5 mesh with 150 ml wash solution (consisting of macroelements and sucrose only of B5 suspension medium). Sieve the suspension through a 0. or poor choice of petioles. Click here to view shaker and suspension cultures Go to next topic Return to Table of Contents Sieving The Suspension 1. some green. 2. An alternative method that will increase embryo numbers is to return the callus on the 0. it should be discarded. 3. small proembryo clusters. The suspension after 7 days will contain large clumps of callus. 2.224 mm mesh and spread thinly and evenly on another disk of 0.5 mm Nitex mesh and then through 0. and elongated single cells.224 mm mesh on top of BOi2Y development medium (this aids in subsequent transfer).5 mm mesh. globular somatic embryos.appearance. Subculturing the suspension may be done provided the large callus clumps are included in the transfer.2 g of cells collected on the 0. Scoop about 1.5 mesh to fresh B5 suspension medium.224 mm mesh in succession.
If the dry embryos are to be germinated on nutrient medium. During this period the somatic embryo accumulates dry weight. 3. ABA induces the expression of desiccation tolerance in the somatic embryos.. torpedo and cotyledonary stages. 3. this is especially critical if there are 500-1000 embryos on a Petri plate. Transfer mesh to BOi2Y maturation I medium. During this time the somatic embryos appear initially as green dots that enlarge as the embryo develops through the globular. and 35 µmol m-2s-1 PPFD for 7-10 days. 2. filter paper or blotting paper to absorb excess moisture. Incubate plates of somatic embryos spread on BOi2Y development medium at 25°C. Spread the loose embryos in a thin layer in a sterile Petri plate. Transfer the embryos to a sterile piece of germination paper. 2. green embryos can be dried and remain viable. During this stage. Wash the somatic embryos from the screen with sterile (if desired) de-ionized water. and 75 µmol m-2s-1 PPFD for 10 days. In other cases. Fully mature embryos will lose their green colour and become yellow-brown. Click here to view globular heart torpedo and cotylendary stages of embryo development Go to next topic Return to Table of Contents Drying 1.Click here to see the setup for sieving Go to next topic Return to Table of Contents Development and Maturation 1. Transfer mesh to BOi2Y maturation medium II. Best results are often achieved if the embryos are transferred to fresh medium every 2-3 days. Do not seal the plates. The ratio of number of developing embryos to medium volume is critical because of competition for nutrients. 16 h photoperiod. it is essential that they remain sterile during drying. Incubate as above for 3-5 days. Large batches of embryos may dry too slowly and lose viability or desiccation tolerance as a result. however. starch and storage proteins. 16 h photoperiod. Incubate plates at 25C. Place in air for 2 days. The rate of drying is critical. heart. leave open or secure the top loosely with two pieces of labelling tape. slow drying through a sequence of progressively lower relative humidities aids in the .
Click here to view a close-up of dry somatic embryos Click here to view a comparison of alfalfa seeds and dry somatic embryos Go to next topic Return to Table of Contents Germination 1. This is the last topic in the methods section. Immature embryos or embryos with minimal desiccation tolerance however will lose viability with time in storage. To germinate. They lack the testa and endosperm associated with true seeds. 2. Click here to view an alfalfa seedling from a dry somatic embryo Click here to review list of References Return to Table of Contents Additional information References The following are selected references that will provide more detail about the development of this somatic embryogenesis system and about somatic embryo development in alfalfa Anandarajah. and B.D. An alternative is to place the dry embryo on moist filter paper as in standard seed germination. store the embryos in a desiccator in a dark. but this varies from batch to batch. After drying is complete. or the embryo can be planted directly into a peat plug or soil in the greenhouse. Enhanced vigour of dry somatic embryos of Medicago sativa L. McKersie. 1990. Dry somatic embryos weigh approximately 1-2 mg each. and only have rudimentary cotyledons. place the dry embryos on agar medium containing 1/2 MS salts and 1% sucrose (if they were kept sterile during drying). Plant Science 71:261-266.acquisition of desiccation tolerance. K. Embryos have remained viable in this state for over two years. 4. 3. with increased sucrose. cool place (in cold room) over a saturated K2CO3 solution to control relative humidity. Direct greenhouse planting usually reduces emergence by about 50%. presumably because it allows immature embryos to complete their maturation. .
B. Somatic embryogenesis in alfalfa. O. Regulation of storage protein synthesis by nitrogen and sulfur nutrients in alfalfa (Medicago sativa L. and G. B. Lai. IN Seed Development and Germination.-M. Plant Sci. 1993. Lai. Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L. 87:69-77. McKersie. McKersie. Effect of nutrition on maturation of alfalfa (Medicago sativa L. 1990. Lai. McKersie. Res. McKersie. 145:507-513. 1994. 73:131-137. Res. Mobilization of Storage Reserves. Germination and Conversion of Alfalfa (Medicago sativa L. 91:87-85 Lai. C.F. S. 2:133-140. McKersie and D. C. somatic embryos.. 3:231-246. Seed Sci. Anandarajah. F. T. Maturation of alfalfa (Medicago sativa L. and B. Plant Physiol. Seed Sci.. Anandarajah.Anandarajah. 1992. A model for the development of dry artificial seed technology. Plant Cell Reports.A. 1994.G. Lecouteux. and B. Senaratna and B.D. Lai.. J.D. Plant Sci.) somatic embryos by absisic acid. 100:211-219. 43:11991202.A. Glutamine enhances storage protein synthesis in Medicago sativa L. The influence of plating density. McKersie. Lai.D. and B.R. 1992.D. Bowley. McKersie. J.833-846. K.W. McKersie and T. Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. Requirement of ethylene for growth of callus and somatic embryogenesis in Medicago sativa L. McKersie.D.) Seeds and Desiccated Somatic Embryos.) somatic embryos. 1992. J. -M. Field Evaluation following two cycles of backcross transfer of somatic embryogenesis to commercial alfalfa germplasm. F. Leprince.-M. McKersie. sucrose and chilling stress.-M. somatic embryos after desiccation.D.. Hendry.. heat shock and abscisic acid.. Chap. Senaratna. 1994. J. and B. McKersie. K. sucrose and light during development on the germination and vigour of Medicago sativa L. Exptl Bot.-M. with sucrose. .D.D. Plant Sci. The mechanisms of desiccation tolerance in developing seeds.. and B.D. 9:451-455.D. Plant Science. Kepczynski.D. J Kigel and G Galili (eds). and B.) somatic embryos.) I Subculture and indirect secondary somatic embryogenesis. Lai. NY. Marcel Dekker. 94:207-213. Brown.C. and B. B. Regulation of starch accumulation in alfalfa (Medicago sativa L. -M. F. F. 1995. Plant Sci. I. F.) somatic embryos. G. Kielly. B. 1993. Lecouteax. and B..D. McKersie. -M. F. K.D. McKersie. 1993. 31: pp. 1993. Plant Cell Tissue Organ Culture 37:151-158. F. Can. 103:209-221. Plant Sci. 1995.
Proc.D. 1992. 1989.129-150. 30. CRC Press. Biol.D. 5-9. McKersie. and B.R. MN.: The effect of polyamines and their biosynthetic precursors on somatic embryogenesis in Medicago sativa L. 1993. Chapter 14: 231-255. Artificial seeds of alfalfa: induction of desiccation tolerance in somatic embryos. Synthetic Seeds of Alfalfa.McKersie. Brown and J. McKersie. McKersie and S. S.D. Shetty. Biol.Sc.) SpringerVerlag pp.152-169. Shargool and T.. A comparison of desiccation tolerance in zygotic and somatic embryos of alfalfa (Medicago sativa). and F. Van Acker.Sc. Lai. Ph. 1989 Susan Van Acker.D. B. 1994. Application of artificial seed technology in the production of hybrid alfalfa (Medicago sativa L.D. Bewley.. 1993. 1994.D. Bowley. M. YPS Bajaj (ed. B. 1993.D. Plant Science 65:253-259. McKersie. IN: K. B. Drying Somatic Embryos for Use as Artificial Seed.C. B. M. Induction and utilization of somatic embryos of alfalfa (Medicago sativa). thioproline and potassium mediated stimulation of somatic embryogenesis in alfalfa (Medicago sativa L). Van Acker. T.D. and S. 26:85-90. Theses on alfalfa somatic embryogenesis at Crop Science. Bowley. Fang-ming Lai.R. B. B. S. Maturation and conversion of alfalfa (Medicago sativa) somatic embryos.McKersie.) Synseeds: Application of Synthetic Seeds to Crop Improvement. Proline. University of Guelph Karin Schneider. Maturation and Desiccation of Somatic Embryos IN: Biotechnology in Agriculture and Forestry Vol. Redenbaugh (ed. M.) somatic embryos. D.. Desiccation tolerance of alfalfa (Medicago sativa L.D. 1990.. T. and B.D. T. St. 1990.D. Senaratna and S.R. Barron Mertens. Influence of Abscisic acid. Ngo eds. T. 1989. McKersie. Aug. P. Yangling Zhang. Bowley. stress pretreatments and drying rates. K. McKersie and S. S. In Vitro Cell Develop. CRC Press pp. IN: Biotechnological Applications of Plant Culture.W. 1992. 17:199-207. In Vitro Cell and Develop. Senaratna. 25:1183-1188. Induction and maturation of alfalfa somatic embryos. of the Plant Growth Regulator Society.). M.T. Desiccation tolerance in somatic embryos..Sc. 1994.R. Bowley. Senaratna. Bowley.R. Senaratna. Paul. .Sc. Plant Science 88: 185193.
Induction of somatic embryogenesis in suspension cultures of Medicago sativa.2 30000 6000 Return to Table of Contents View another media composition .4-D Kinetin Sucrose Agar pH 5.5 5 1 0.Kasia Napierala. Macronutrients NH4H2PO4 KNO3 CaCl2 2H2O MgSO4 7H2O K2SO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na2MoO4 2H2O CuSo4 5H2O CoCl2 6H2O Na2 EDTA FeSO4 7H2O Amino Acids Proline Thioproline Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other 2.8 300 2500 200 400 4350 1 5 10 1 0. M.1 0. Return to Table of Contents Media Composition SH Induction Medium Components are given in mg/L. 1995.2 0.Sc.1 20 15 288 53 200 5 0.
8 1.4 1.025 0.025 43 100 1 1 10 1 20000 5.4-D Sucrose pH 2500 150 250 134 150 0.5 Return to Table of Contents View another media composition BOi2Y development medium Components are given in mg/L Macronutrients NH4NO3 KCl KNO3 Ca(NO3)2 MgSO4 7H2O KH2PO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O 1000 65 1000 347 35 300 0.5 .6 4.B5 Suspension Medium Components are given in mg/L Macronutrients KNO3 CaCl2 2H2O MgSO4 7H2O (NH4)2 SO4 NaH2PO4 H2O Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na2MoO4 2H2O CuSo4 5H2O CoCl2 6H2O Na Fe EDTA Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other 2.25 0.75 3 10 2 0.
1 0.5 0.8 Return to Table of Contents View another media composition BOi2Y Maturation I medium Components are given in mg/L Macronutrients NH4NO3 1000 KCl 65 KNO3 1000 Ca(NO3)2 347 MgSO4 7H2O 35 KH2PO4 300 K2 SO4 4350 Micronutrients KI 0.6 MnSO4 H2O 4.4 ZnSO4 7H2O 1.5 Na Fe EDTA 32 Amino Acids Glycine 2 Vitamins Myo-inositol 100 .1 50000 2000 6000 5.8 H3BO3 1.Na Fe EDTA Amino Acids Glycine Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other Sucrose Yeast Extract Agar pH 32 2 100 0.
5 32 2 100 0.8 Return to Table of Contents View another media composition BOi2Y Maturation II medium Components are given in mg/L Macronutrients NH4NO3 KCl KNO3 Ca(NO3)2 MgSO4 7H2O KH2PO4 Micronutrients KI H3BO3 MnSO4 H2O ZnSO4 7H2O Na Fe EDTA Amino Acids Glycine Vitamins Myo-inositol Nictonic acid Pyridoxine HCl Thiamine HCL Other ABA Sucrose Yeast Extract Agar pH 1000 65 1000 347 35 300 0.1 Other Sucrose 50000 Yeast Extract 2000 Agar 6000 pH 5.6 4.3 50000 2000 6000 5.1 0.4 1.1 5.Nictonic acid 0.8 Return to Table of Contents .1 Thiamine HCL 0.5 Pyridoxine HCl 0.5 0.8 1.
View another media composition Composition of Media used for alfalfa somatic embryogenesis SH induction medium B5 suspension medium Development medium Maturation phase I medium Maturation phase II medium .
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